Oligoribonucleotides and ribonucleases for cleaving rna

ABSTRACT

Oligomeric compounds including oligoribonucleotides and oligoribonucleosides are provided that have subsequences of 2′-pentoribofuranosyl nucleosides that activate dsRNase. The oligoribonucleotides and oligoribonucleosides can include substituent groups for increasing binding affinity to complementary nucleic acid strand as well as substituent groups for increasing nuclease resistance. The oligomeric compounds are useful for diagnostics and other research purposes, for modulating the expression of a protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to oligonucleotide therapeutics. Also included in the invention are mammalian ribonucleases, i.e., enzymes that degrade RNA, and substrates for such ribonucleases. Such a ribonuclease is referred to herein as a dsRNase, wherein “ds” indicates the RNase&#39;s specificity for certain double-stranded RNA substrates. The artificial substrates for the dsRNases described herein are useful in preparing affinity matrices for purifying mammalian ribonuclease as well as non-degradative RNA-binding proteins.

CROSS REFERENCED TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 10/078,949, filedFeb. 20, 2002, which is a continuation of U.S. Ser. No. 09/479,783,filed Jan. 1, 2000, now abandoned, which is a divisional of U.S. Ser.No. 08/870,608, filed Jun. 6, 1997, now U.S. Pat. No. 6,107,094, whichis a continuation in part of U.S. Ser. No. 08/659,440 filed Jun. 6,1996, now U.S. Pat. No. 5,898,031.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledISIS5030USC1SEQ.txt, created on Jul. 13, 2009 which is 2 KB in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention is directed to the synthesis and use of oligomericcompounds, including oligoribonucleotides and oligoribonucleosides,useful for strand cleavage of target RNA strands. Included in theinvention are oligoribonucleotides having modified sugars, bases orphosphate backbones and oligoribonucleosides having standard sugars andbases or modified sugars and bases linked together via non-phosphatebackbones. Further included in the invention are chimericoligoribonucleotides and oligoribonucleosides having mixed backbones,either phosphate or non-phosphate. Also included in the invention aremammalian ribonucleases, i.e., enzymes that degrade RNA. Such aribonuclease is referred to herein as a dsRNase, wherein “ds” indicatesthe RNase's specificity for certain double-stranded RNA substrates. Theoligoribonucleotides, oligoribonucleosides, ribonucleases andribonuclease substrates of the invention are useful for therapeutics,diagnostics and as research reagents.

BACKGROUND OF THE INVENTION

Oligonucleotides are known to hybridize to single-stranded DNA or RNAmolecules. Hybridization is the sequence-specific base pair hydrogenbonding of nucleobases of the oligo-nucleotides to nucleobases of targetDNA or RNA. Such nucleobase pairs are said to be complementary to oneanother.

The complementarity of oligonucleotides has been used for inhibition ofa number of cellular targets. Such complementary oligonucleotides arecommonly described as being antisense oligonucleotides. Various reviewsdescribing the results of these studies have been published includingProgress In Antisense Oligonucleotide Therapeutics, Crooke, S. T. andBennett, C. F., Annu. Rev. Pharmacol. Toxicol., 1996, 36, 107-[29. Theseoligonucleotides have proven to be very powerful research tools anddiagnostic agents. Further, certain oligonucleotides that have beenshown to be efficacious are currently in human clinical trials.

To date most oligonucleotides studied have been oligodeoxynucleotides.Antisense oligodeoxynucleotides are believed to cause a reduction intarget RNA levels principally through the action of RNase H, anendonuclease that cleaves the RNA strand of DNA:RNA duplexes. Thisenzyme, thought to play a role in DNA replication, has been shown to becapable of cleaving the RNA component of the DNA:RNA duplexes in cellfree systems as well as in Xenopus oocytes. Rnase H is very sensitive tostructural alterations in antisense oligonucleotides. This sensitivityis such that prior attempts to increase the potency of oligonucleotidesby increasing affinity, stability, lipophilicity and othercharacteristics by chemical modifications of the oligonucleotide haveoften resulted in oligonucleotides that are no longer substrates forRnase H. In addition, Rnase H activity is quite variable. Thus a givendisease state may not be a candidate for antisense therapy only becausethe target tissue has insufficient Rnase H activity. Therefore it isclear that effective terminating mechanisms in addition to Rnase H areof great value to the development of therapeutic and other agents.

Several publications describe the interaction of Rnase H andoligonucleotides. A recently publication is: Crooke, et. al., Biochem.J., 1995, 312, 599-608. Other earlier papers are: (1) Dagle et al.,Nucleic Acids Research, 1990, 18, 4751; (2) Dagle et al., AntisenseResearch And Development, 1991, 1, 11; (3) Eder et al., J. Biol. Chem.,1991, 266, 6472; and (4) Dagle et al., Nucleic Acids Research, 1991, 19,1805. According to these publications, DNA oligonucleotides having bothunmodified phosphodiester internucleoside linkages and modifiedphosphorothioate internucleoside linkages are substrates for cellularRNase H. Since they are substrates, they activate the cleavage of targetRNA by RNase H. However, these authors further noted that in Xenopusembryos, both phosphodiester linkages and phosphorothioate linkages arealso subject to exonuclease degradation. Nuclease degradation isdetrimental since it rapidly depletes the oligonucleotide.

As described in references (1), (2) and (4), to stabilizeoligonucleotides against nuclease degradation while still providing forRNase H activation, 2′-deoxy oligonucleotides having a short section ofphosphodiester linked nucleosides positioned between sections ofphosphoramidate, alkyl phosphonate or phosphotriester linkages wereconstructed. While the phosphoramidate-containing oligonucleotides werestabilized against exonucleases, in reference (4) the authors noted thateach phosphoramidate linkage resulted in a loss of 1.6° C. in themeasured T_(m), value of the phosphoramidate-containingoligonucleotides. Such a decrease in the T_(m), value is indicative of adecrease in hybridization between the oligonucleotide and its targetstrand.

Other authors have commented on the effect such a loss of hybridizationbetween an oligonucleotide and its target strand can have.Saison-Behmoaras et al. (EMBO Journal, 1991, 10, 1111) observed thateven though an oligonucleotide could be a substrate for Rnase H,cleavage efficiency by Rnase H was low because of weak hybridization tothe mRNA. The authors also noted that the inclusion of an acridinesubstitution at the 3′ end of the oligonucleotide protected theoligonucleotide from exonucleases.

U.S. Pat. No. 5,013,830, issued May 7, 1991, discloses mixed oligomerscomprising an RNA oligomer, or a derivative thereof, conjugated to a DNAoligomer via a phosphodiester linkage. The RNA oligomers also bear2′-O-alkyl substituents. However, being phosphodiesters, the oligomersare susceptible to nuclease cleavage.

European Patent application 339,842, published Nov. 2, 1989, discloses2′-O-substituted phosphorothioate oligonucleotides, including2′-O-methylribooligonucleotide phosphorothioate derivatives. Theabove-mentioned application also discloses 2′-O-methyl phosphodiesteroligonucleotides which lack nuclease resistance.

U.S. Pat. No. 5,149,797, issued Sep. 22, 1992, discloses mixed phosphatebackbone oligonucleotides which include an internal portion ofdeoxynucleotides linked by phosphodiester linkages, and flanked on eachside by a portion of modified DNA or RNA sequences. The flankingsequences include methyl phosphonate, phosphoromorpholidate,phosphoropiperazidate or phosphoramidate linkages.

U.S. Pat. No. 5,256,775, issued Oct. 26, 1993, describes mixedoligonucleotides that incorporate phosphoramidate linkages andphosphorothioate or phosphorodithioate linkages.

U.S. Pat. No. 5,403,711, issued Apr. 4, 1995, describes RNA:DNA probestargeted to DNA. The probes are labeled and are used in a system thatincludes RNase H. The RNase H enzyme cleaves those probes that bind toDNA targets. The probes can include modified phosphate groups. Mentionedare phosphotriester, hydrogen phosphonates, alkyl or aryl phosphonates,alkyl or aryl phosphoramidates, phosphorothioates or phosphoroselenates.

In contrast to the pharmacological inhibition of gene expression via theRNase H enzyme, it is becoming clear that organisms from bacteria tohumans use endogenous antisense RNA transcripts to alter the stabilityof some target mRNAS and regulate gene expression, see Nellen, W., andLichtenstein, C., Curr. Opin. Cell. Biol., 1993, 18, 419-424 and Nellen,W., et al, Biochem. Soc. Trans. 1992, 20, 750-754. Perhaps one of thebest examples comes from certain bacteria where an antisense RNAregulates the expression of mok mRNA, which is required for thetranslation of the cytotoxic hok protein. Thus as the antisense leveldrops, mok mRNA levels and consequently hok protein levels rise and thecells die, see Gerdes, K. et al., J. Mol. Biol., 1992, 226, 637-649.Other systems regulated by such mechanisms in bacteria include the RNAI-RNA II hybrid of the ColE1 plasmid, see Haeuptle, M. T., Frank, R.,and Dobberstein, B., Nucleic Acids Res. 1986, 14, 1427, Knecht, D., CellMotil. Cytoskel., 1989, 14, 92-102; and Maniak, M., and Nellen, W.,Nucleic Acids Res., 1990, 18, 5375-5380; OOP-cII RNA regulation inbacteriophage Lambda, see Krinke, L., and Wulff, D. L. (1990) GenesDev., 1990, 4, 2223-2233; and the copA-copT hybrids in E. coli. SeeBlomberg, P., Wagner, E. G., and Nordstrom, K., EMBO J., 1990, 9,2331-2340. In E. coli the RNA:RNA duplexes formed have been shown to besubstrates for regulated degradation by the endoribonuclease RNase III.Duplex dependent degradation has also been observed in thearchaebacterium, Halobacterium salinarium, where the antisensetranscript reduces expression of the early (T1) transcript of the phagegene phiH, see Stolt, P., and Zillig, W., Mol. Microbiol., 1993, 7,875-882. In several eukaryotic organisms endogenous antisensetranscripts have also been observed. These include p53, see Khochbin andLawrence, EMBO, 1989, 8, 4107-4114; basic fibroblast growth factor, seeVolk et al, EMBO, 1989, 8, 69, 2983-2988; N-myc, see Krystal, G. W.,Armstrong, B. C., and Battey, J. F., Mol. Cell. Biol., 1990, 10,4180-4191; eIF-2α, see Noguchi et al., J. Biol. Chem., 1994, 269,29161-29167. The conservation of endogenously expressed antisensetranscripts across evolutionary lines suggests that their biologicalroles and molecular mechanisms of action may be similar.

In bacteria, RNase III is the double stranded endoribonuclease (dsRNase)activity responsible for the degradation of some antisense:sense RNAduplexes. RNase III carries out site-specific cleavage ofdsRNA-containing structures, see Saito, H. and Richardson, C. C., Cell,1981, 27, 533-540. The RNase III also plays an important role in mRNAprocessing and in the processing of rRNA precursors into 16S, 23S and 5Sribosomal RNAs, see Dunn, J. J. and Studier, F. W. J. Mol. Biol., 1975,99, 487. In eukaryotes, a yeast gene (RNT1) has recently been clonedthat codes for a protein that has homology to E. coli RNase III andshows dsRNase activity in ribosomal RNA processing, see Elela, S. A.,Igel, H. and Ares, M. Cell, 1996, 85, 115-124. Avian cells treated withinterferon produce and secrete a soluble nuclease capable of degradingdsRNA, see Meegan, J. and Marcus, P. I., Science, 1989, 244, 1089-1091.However such a secreted dsRNA activity is not a likely candidate to beinvolved in cytoplasmic degradation of antisense:sense RNA duplexes.Despite these findings almost nothing is known about human or mammaliandsRNAse activities. While it has been recognized that regulation (viaany mechanism) of a target RNA strand would be useful, to date only twomechanisms for eliciting such an effect are known. These arehybridization arrest and use of an oligodeoxynucleotide to effect RNaseH cleavage of the RNA target. Accordingly, there remains a continuinglong-felt need for methods and compounds for regulation of target RNA.Such regulation of target RNA would be useful for therapeutic purposesboth in vivo and ex vivo and, as well as, for diagnostic reagents and asresearch reagents including reagents for the study of both cellular andin vitro events.

SUMMARY OF THE INVENTION

In accordance with this invention there are provided oligomericcompounds formed from a linear sequence of linked ribonucleosidesubunits that are specifically hybridizable to a preselected RNA target.The oligomeric compounds have at least a first segment and a secondsegment. The first segment incorporates at least one ribonucleosidesubunit that is modified to improve at least one of its pharmacokineticproperties, its binding characteristics to target RNA or to modify itscharge. The second segment includes at least four consecutiveribofuranosyl nucleoside subunits. The subunits of the oligomericcompounds are connected together in a linear sequence by internucleosidelinkages that are stabilized from degradation as compared tophosphodiester linkages.

In certain preferred embodiments of the invention, the compounds willinclude a third segment having properties corresponding to theproperties of the first segment. It is preferred to position the secondsegment between the first and third segments such that they form acontinuous, linear sequences of linked nucleoside units. In preferredcompounds the number of such linked nucleoside subunits will range fromabout eight to about fifty with a more preferred range being from abouttwelve to about thirty linked nucleoside subunits.

Modification of pharmacokinetic properties includes any one or more ofthe modification of binding, absorption, distribution or clearanceproperties of the compound. Modification of binding characteristicsincludes modification of the affinity or specificity of said compound toits target RNA. Modification of the charge of said compound includesmodifying the net charge of the compound as compared to an unmodifiedcompound. Normally modification of charge will decrease the overall netnegative charge of a phosphorus linked oligomeric compound to providethe compound with less negative charge, a neutral charge or a netpositive charge.

Further in accordance with this invention, there are provided oligomericcompounds formed from linear sequences of linked ribonucleoside subunitsthat are specifically hybridizable to a preselected RNA target. Theoligomeric compounds have at least a first segment and a second segment.The first segment incorporates at least one ribonucleoside subunit thatis functionalized to provide greater affinity to the target RNA. Thesecond segment includes at least four ribofuranosyl nucleoside subunits.The subunits of the oligomeric compounds are connected together in alinear sequence by internucleoside linkages that are modified tostabilize the linkages from degradation as compared to phosphodiesterlinkages.

In certain preferred oligomeric compounds of the invention, the first orfirst and third segments of oligomeric compounds are formed ofnucleoside subunits that include 2′-substituent groups thereon. Inpreferred embodiments, the 2′-substituent group includes fluoro, C₁-C₂₀alkoxy, C₁-C₉ aminoalkoxy, allyloxy, imidazolylalkoxy and polyethyleneglycol. Preferred alkoxy substituents include methoxy, ethoxy andpropoxy. A preferred aminoalkoxy substituent is aminopropoxy. Apreferred imidazolylalkoxy substituent is imidazolylpropoxy. A preferredpolyethylene glycol substituent is —O-ethyl-O-methyl, i.e.,methoxyethoxy or —O—CH₂—CH₂—O—CH₃.

In further preferred oligomeric compounds of the invention, theoligomeric compounds are formed of nucleoside subunits that are modifiedby including certain selected nucleobases thereon. In preferredembodiments, the selected nucleobases include 2,6-diaminopurine,N2-alkylpurines, N2-aminoalkylpurines, 7-deaza-7-substituted purines,5-substituted pyrimidines, and 2-substituted pyrimidines.

Other preferred oligomeric compounds of the invention includeoligoribonucleotides having nucleoside subunits connected by phosphoruslinkages including phosphorothioate, 3′-(or -5′)deoxy-3′-(or -5′)thio-phosphorothioate, phosphoro-dithioate, phosphoroselenates, 3′-(or-5′)deoxy phosphinates, borano phosphates, 3′-(or -5′)deoxy-3′-(or5′-)amino phosphor-amidates, hydrogen phosphonates, borano phosphateesters, phosphoramidates, alkyl or aryl phosphonates andphospho-triester linkages. A selected group of oligoribonucleotidelinkages for use in linking the nucleosides of the second segmentinclude phosphorothioate, phosphinates and phosphor-amidates, all ofwhich are charged species.

Further preferred oligomeric compounds of the invention may also includeoligoribonucleotides having nucleoside subunits connected by carbonate,carbamate, silyl, sulfur, sulfonate, sulfonamide, formacetal,thioformacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethyliminolinkages.

Further preferred oligomeric compounds of the invention include havingnucleoside subunits connected by alternating phosphorus andnon-phosphorous linkages. Such non-phosphorous linkages includecarbonate, carbamate, silyl, sulfur, sulfonate, sulfonamide, formacetal,thioformacetal, oxime, methyleneimino, methylenemethylimino,methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethyliminolinkages while the phosphorous linkages include phosphodiester,phosphorothioate, 3′-(or -5′)deoxy-3′-(or -5′)thio-phosphorothioate,phosphorodithioate, phosphoroselenates, 3′-(or -5′)deoxy phosphinates,borano phosphates, 3′-(or -5′)deoxy-3′-(or 5′-)amino phosphoramidates,hydrogen phosphonates, borano phosphate esters, phosphoramidates, alkylor aryl phosphonates and phosphotriester linkages.

Further preferred oligomeric compounds of the invention includeoligoribonucleotides, oligoribonucleosides or mixtures ofoligoribonucleotides and oligoribonucleosides having a plurality oflinked nucleoside subunits that are linked in a sequences that iscomplementary strand of target RNA and wherein the sequence of thecompound is divided into a first subsequence or segment and a secondsubsequence or segment. The first subsequence comprises linkednucleoside subunits bearing 2′-O-substituted-pentofuranosyl sugarmoieties and the second subsequence comprises linked nucleoside subunitsbearing 2′-hydroxyl-pentofuranosyl sugar moieties. Preferably, saidsecond subsequence has from four to twelve or more nucleoside subunits,and more preferably, has five to about nine nucleoside subunits. Infurther preferred embodiments there exists a third subsequence, thenucleoside subunits of which are selected from those which areselectable for the first subsequence. It is preferred that the secondsubsequence be positioned between the first and the third subsequences.Such oligomeric compounds of the invention are also referred to as“chimeras,” “chimeric” or “gapped” oligoribonucleotides oroligoribonucleosides.

In further preferred oligomeric compounds of the invention, nucleosidesubunits bearing substituents that are modified to improve at least oneof: pharmacokinetic binding, absorption, distribution or clearanceproperties of the compound: affinity or specificity of said compound tosaid target RNA: or modification of the charge of said compound,compared to an unmodified compound; are located at one or both of the 3′or the 5′ termini of the oligomeric compounds. In certain preferredcompounds there are from one to about eight nucleoside subunits that aresubstituted with such substituent groups.

The nucleoside subunits are joined together in a linear sequence to formthe oligomeric compounds of the invention. Each nucleoside subunitincludes a base fragment and a sugar fragment. The base fragmentcomprises a heterocyclic base, alternately hereinafter referred to as anucleobase. The bases or nucleobases are covalently bonded to the sugarfragment. The sugar fragments may include a 2′-substituted sugar moiety,a 2′-hydroxyl sugar moiety or a sugar surrogate moiety.

Preferred nucleobases of the invention include purines and pyrimidinessuch as adenine, guanine, cytosine, uridine, and thymine, as well asother synthetic and natural nucleobases such as xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines andguanines, 5-trifluoromethyl and other 5-substituted uracils andcytosines, and 7-methylguanine. Further purines and pyrimidines includethose disclosed in U.S. Pat. Nos. 3,687,808, 5,484,908, 5,459,255,5,457,191 and 5,614,617 (corresponding to U.S. patent application Ser.No. 07/971,978), and those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Preferred sugar fragments are pentoribofuranosyl sugar moieties, i.e.,the “natural” sugar moiety of messenger ribonucleic acids. Othersugar-like or sugar surrogate compounds suitable for use in theoligoribonucleotides or oligoribonucleosides of the invention includecyclobutyl nucleoside surrogates as described in U.S. Pat. No.5,359,044, pyrrolidine nucleoside surrogates as described in U.S. Pat.No. 5,519,134, morpholino nucleoside surrogates as described in U.S.Pat. Nos. 5,142,047 and 5,235,033, and in related patent disclosures,and PNA (peptide nucleic acid) nucleoside surrogates.

In further preferred embodiments of the invention there are providedsynthetic oligomeric compounds that are specifically hybridizable with apreselected RNA target and where the compounds include a first segmentincluding at least one surrogate nucleoside subunit and a second segmentcomprising at least four ribofuranosyl nucleoside subunits located in aconsecutive sequence and having 2′-hydroxyl moieties thereon. Furtherthe nucleoside subunits of the oligomeric compound are connected byinternucleoside linkages that are stable to degradation as compared tophosphodiester bonds.

In other preferred embodiments of the invention, there are providedsynthetic oligomeric compounds that are specifically hybridizable with apreselected RNA target and that include a first segment having at leastone ribofuranosyl nucleoside subunit that is not a DNA or RNA “major”building block nucleoside and a second segment that includes at leastfour consecutive ribofuranosyl nucleoside subunits having 2′-hydroxylmoieties thereon. The nucleoside subunits of the compounds are connectedby internucleoside linkages which are modified to stabilize the linkagesfrom degradation as compared to phosphodiester linkages. Nucleosidesubunits that are not DNA or RNA major building block nucleosides asthat term is used in connection with this invention, are members of thegroup consisting of adenosine, 2′-deoxyadenosine, guanosine,2′-deoxyguanosine, cytidine, 2′-deoxycytidine, uridine and2′-deoxythymidine. As such, this group excludes “minor” nucleosides thatmay be found in tRNA or in other nucleic acids.

The invention also provides methods of for specifically cleavingpreselected RNA. These methods include contacting the RNA with acompound that includes at least twelve ribofuranosyl nucleosidessubunits joined in a sequence which is specifically hybridizable withthe preselected RNA. The nucleoside subunits are joined byinternucleoside bonds that are stable to degradation as compared tophosphodiester bonds. The compound has at least one segment thatincludes at least one modified nucleoside subunit, which modifiednucleoside subunit is modified to improve at least one ofpharmacokinetic binding, absorption, distribution or clearanceproperties of the compound; affinity or specificity of the compound totarget RNA; or modification of the charge of the compound, compared toan unmodified compound. The compound additionally includes a furthersegment having at least four ribonucleoside subunits.

The invention also provides methods for treating an organism having adisease characterized by the undesired production of a protein. Thesemethods include contacting the organism with an oligomeric compound ofthe invention having a sequence of nucleoside subunits capable ofspecifically hybridizing with a complementary strand of ribonucleic acidwith at least one of the nucleoside subunits being functionalized tomodify one of more properties of the oligomeric compounds compared tonative RNA. The compound further includes a plurality of the nucleosidesubunits having 2′-hydroxyl-pentofuranosyl sugar moieties.

Further in accordance with the present invention, there are providedcompositions including a pharmaceutically effective amount of anoligomeric compound having a sequence of nucleoside subunits capable ofspecifically hybridizing with a complementary strand of RNA and whereinat least one of the nucleoside subunits is modified to improve at leastone of pharmacokinetic binding, absorption, distribution or clearanceproperties of the compound; affinity or specificity of said compound tosaid target RNA; or modification of the charge of said compound,compared to an unmodified compound. In such compounds, a plurality ofthe nucleoside subunits have 2′-hydroxyl-pentofuranosyl sugar moieties.The compositions further include a pharmaceutically acceptable diluentor carrier.

The present invention also provides mammalian ribonucleases, isolatablefrom human T24 cells, other cell lines, and rat tissues, that degradeRNA in an oligoribonucleotide:RNA duplex. Such a ribonuclease isreferred to herein as a dsRNase, wherein “ds” indicates the RNase'sspecificity for certain double-stranded RNA substrates. Usefulsubstrates for such dsRNases are also herein provided, as well asaffinity matrices comprising such substrates.

Methods are also provided for in vitro modification of asequence-specific target RNA including contacting a test solutioncontaining a dsRNase enzyme, i.e., a double stranded RNase enzyme, andsaid target RNA with an oligomeric compound. The oligomeric compound hasa sequence of nucleoside subunits capable of specifically hybridizing toa complementary strand of the nucleic acid, where at least one of thenucleoside subunits is functionalized to increase the binding affinityor binding specificity of the oligoribonucleotide to the complementarystrand of nucleic acid, and where a plurality of the nucleoside subunitshave 2′-hydroxyl-pentofuranosyl sugar moieties.

There are also provided methods of concurrently enhancing hybridizationand/or dsRNase enzyme activation in an organism that includes contactingthe organism with an oligomeric compound having a sequence of nucleosidesubunits capable of specifically hybridizing to a complementary strandof target RNA. At least one of the nucleoside subunits is modified toimprove at least one of pharmacokinetic binding, absorption,distribution or clearance properties of the compound; affinity orspecificity of said compound to said target RNA; or modification of thecharge of said compound, compared to an unmodified compound. Again, aplurality of the nucleoside subunits have 2′-hydroxy-pentofuranosylsugar moieties.

The invention further provides diagnostic methods for detecting thepresence or absence of abnormal RNA molecules, or abnormal orinappropriate expression of normal RNA molecules in organisms or cells.The invention further provides research reagents for modulating enzymeactivity including dsRNase activity in in vitro solutions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically depicts certain illustrative chimeric oligomericcompounds of the invention wherein open squares represent 2′-methoxymodified ribonucleotides, filled circles represent 2′-hydroxylribonucleotides and phosphorothioate linkages are utilized through thecompounds shown in the figure.

FIG. 2 depicts Ha-ras mRNA levels in cells treated with full 2′-methoxyor chimeric RNA gapmer oligonucleotides. Northern blot analyses forHa-ras mRNA levels in T24 cells treated with the indicated doses of full2″-methoxy oligonucleotide (panel 2A) or 3 gap oligoribonucleotide(panel 2C) for 24 hrs. are shown. The upper band is the signal forHa-ras, this signal was normalized to that obtained for G3PDH (lowerband), relative Ha-ras levels were determined and are presentedgraphically (panels 2C-2D). Neither oligonucleotide treatment reducedHa-ras mRNA levels.

FIG. 3 shows Northern blot analyses of T24 cell treated as in FIG. 2except with chimeric RNA gapmer oligonucleotides containing either a 5,7 or 9 ribonucleotide gap or a full ribonucleotide molecule (left panels3A, 3B, 3C and 3D, respectively); cells were also treated with a controloligoribonucleotide that contains four mismatched base pairs to theHa-ras mRNA sequence (left panel 3E). Ha-ras signals were normalized tothat of G3PDH and relative Ha-ras levels are shown graphically (rightpanels).

In FIG. 4, the effect of T24 cytosolic extracts and RNase H on duplexesin vitro are shown. A 17 base pair duplex consisting of the Ha-rastargeted 9 RNA gapmer oligonucleotide annealed to a ³²P-labeled RNAcomplement was incubated with 3 ug of T24 cytosolic protein fraction forthe indicated times at 37° C., the reaction was stopped and productswere resolved on a denaturing polacrylamide gel. Digestion products(arrows) indicate that cleavage of the duplex is restricted to theRNA:RNA region (see schematic of duplex, far right).

FIG. 5 shows the same 9 RNA gapmer oligonucleotide:RNA duplex as in FIG.4, incubated with or without E. coli RNase H (− and +, respectively).The lack of digestion products indicates that this duplex is not asubstrate for RNase H. Duplexes consisting of ³²P-labeled RNA annealedto either a full oligodeoxynucleotide (middle panel) or 9 DNA gapmeroligonucleotide (left panel) are substrates for cleavage by RNase H andthus generate digestion products as expected (arrows).

FIG. 6 depicts SDS-polyacrylamide gel electrophoretic analysis of theconcentrated rat liver active fractions after size exclusionchromatography. MW, molecular weight markers in kilodaltons (kD).Fraction 3 (lane 4), having an apparent molecular weight in the range ofabout 35 to about 100 kD, with much of the material having an apparentmolecular weight in the range 50 to about 80 kD, had the greatest amountof dsRNase activity.

FIG. 7 shows analysis of products of digestion of dsRNAse substrates bynative polyacrylamide gel electrophoresis. Antisense and senseoligonucleotides were preannealed and incubated with cellular extractsand purified dsRNases as described herein. Lane 1, untreated “sense”strand RNA; lane 2, “sense” strand RNA treated with 0.02 units RNase V1;remaining lanes: dsRNAse substrates treated with 0.02 (lane 3) and 0.002(lane 4) units of RNase V1, with unpurified nuclear extract for 0minutes (lane 5) or 240 minutes (lane 6), with unpurified nuclearextract for 240 minutes without Mg⁺⁺ (lane 7), with unpurified cytosolicextract for 240 minutes (lane 8), with ion exchange purified cytosolicextract for 240 minutes in the presence (lane 9) or absence (lane 10) ofMg⁺⁺, and with ion exchange/gel filtration purified cytosolic extractfor 240 minutes in the presence (lane 9) or absence (lane 10) of Mg⁺⁺.

FIG. 8 shows analysis of products of digestion of dsRNAse substrates bydenaturing polyacrylamide gel electrophoresis. Lane 1, “sense” strandRNA treated with 5×10 units of RNase A; lane 2, “sense” strand RNAtreated with 0.02 units RNase V1; lanes 3-9: dsRNAse products treatedwith 0.02 (lane 3) and 0.002 (lane 4) units of RNase V1, with unpurifiednuclear extract for 0 minutes (lane 5) or 240 minutes (lane 6), withunpurified cytosolic extract for 240 minutes (lane 7), with ion exchangepurified cytosolic extract for 240 minutes (lane 8), and with ionexchange/gel filtration purified cytosolic extract for 240 minutes (lane9). Lane 10, base hydrolysis ladder.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

While not wishing to be bound by theory, it is now believed that by theuse of certain chemically modified oligomeric compounds, one can exploitcertain enzymatic activities in eukaryotic cells, including human cells,resulting from the unexpected interaction of these compounds with atarget RNA strand to form double-stranded RNA like structures that arecleaved by certain enzymes. Heretofore, such activity has not recognizednor exploited in eukaryotic systems. It has now been found that theoligomeric compounds of the invention have certain RNA like featuresthat allow them to form a double stranded structure with a targeted RNAregion and this double stranded structure is subsequently degraded byeukaryotic dsRNases, i.e. double-stranded RNase enzymes, in a cell ortest solution. Using T24 human bladder carcinoma cells as anillustrative eukaryotic cellular system, it has been demonstrated thatthis activity is present at comparable levels in both the nuclear andcytoplasmic fractions.

In certain illustrative procedures provided herein to illustrate thisinvention, in common with some other known nuclease activities, it hasbeen found that this activity leaves 5′ phosphate and 3′ hydroxyl groupsafter cleavage of the RNA substrate. This generation of 5′ phosphate, 3′hydroxyl termini is a feature in common with several other nucleasesthat recognize double-stranded nucleic acid molecules, including RNaseHI and II that cleave the RNA component of a DNA:RNA duplex in E. coli.,RNase III which catalyses the hydrolysis of high molecular weight doublestranded RNA and mediates degradation of sense-antisense duplexes, andRNase V1.

Many components of mRNA degradation systems have been conserved betweenprokaryotes and eukaryotes. It has now been found that like prokaryoticorganisms, in which RNase III carries out the degradation ofsense-antisense hybrids to regulate expression of some genes, humancells have conserved an activity capable of performing a similar role.In addition to other uses including therapeutic and diagnostic uses, byvirtue of this activity the compounds of this invention can be used asresearch reagents to assist in understanding how human cells useendogenously expressed antisense transcripts to modulate geneexpression.

The vast majority of antisense oligonucleotides used experimentally orcurrently being tested in the clinic in humans are modifiedoligodeoxynucleotides. It has been demonstrated that the heteroduplexformed between such oligodeoxynucleotide antisense compounds and theirtarget RNA is recognized by an intracellular nuclease, RNase H, thatcleaves only the RNA strand of this duplex. Although RNase H mediateddegradation of target RNA has proven a useful mechanism, it has certainlimitations. RNase H is highly sensitive to structural modificationsmade to the antisense oligonucleotides and thus most of themodifications designed to improve the therapeutic properties such asincreased affinity, increased nuclease resistance and greater cellularpermeability have resulted in oligonucleotides that do not supportcleavage by RNase H. Another limitation to RNase H as a terminatingmechanism of antisense action is the fact that the oligonucleotides mustbe DNA ‘like’, and in being DNA ‘like’, such oligonucleotides haveinherently low affinity to their target RNA. Strategies designed tocircumvent this low affinity include the design of “gapmer”oligonucleotides that are composed of a stretch of high affinitychemically modified oligonucleotides on the 5′ and 3′ ends (the wings)with a stretch of unmodified deoxyoligonucleotides in the center (thegap). DNA gapmers, i.e., oligodeoxynucleotides gapmers, havesignificantly higher affinities for their target thanoligodeoxynucleotides, however, depending on the size of the DNA gap,RNase H activity has been shown to be compromised.

In using RNase H as a termination mechanism via RNA degradation, thecellular localization and tissue distribution of RNase H must also beconsidered. RNase H activity is primarily localized to the nucleusalthough it has been detected in the cytoplasm at lower levels. Most ofa given mRNA is found in the cytoplasm of cells, therefore the idealactivity to be exploited as a terminating mechanism would be one withhigh levels in both the nucleus and the cytoplasm. RNase H activity alsois highly variable from cell line to cell line or between tissues, thusa given disease state may not be a good candidate for RNA degradationonly because the target tissue has insufficient RNase H activity. It isclear that alternative terminating mechanisms for degrading target RNAare highly desirable.

Among other uses, the activity that has now been recognized can now beexploited as an alternative terminating mechanism to RNase H forantisense therapeutics. It has been found that in using RNA-likeoligonucleotides that have high affinity for their target and thushigher potency than DNA-like oligonucleotides, activity can be expressedin human cells. The presence of the activity in both the cytoplasm andthe nucleus allows the compounds of the invention to be used to inhibitmany RNA processing events from nuclear pre-mRNA splicing and transportto degradation of mature transcript in the cytoplasm. To illustrate thisinvention and to compare it to other known antisense mechanisms, e.g.RNase H, the dsRNAse activity induced by the compounds of the inventionhas been examined by targeting it to codon 12 of Ha-ras. As described inU.S. Pat. No. 5,297,248, corresponding to Ser. No. 08/297,248, and itsrelated application International Publication Number WO 92/22651,published Dec. 23, 1992, both commonly assigned with this application,the entire contents of which are herein incorporated by reference, theras oncogenes are members of a gene family that encode related proteinsthat are localized to the inner face of the plasma membrane and havebeen shown to be highly conserved at the amino acid level, to bind GTPwith high affinity and specificity, and to possess GTPase activity.Although the cellular function of ras gene products is unknown, theirbiochemical properties, along with their significant sequence homologywith a class of signal-transducing proteins, known as GTP bindingproteins, or G proteins, suggest that ras gene products play afundamental role in basic cellular regulatory functions related to thetransduction of extracellular signals across plasma membranes.

Three ras genes, designated H-ras, K-ras, and N-ras, have beenidentified in the mammalian genome. Mammalian ras genes acquiretransformation-inducing properties by single point mutations withintheir coding sequences. Mutations in naturally occurring ras oncogeneshave been localized to codons 12, 13, and 61. The most commonly detectedactivating ras mutation found in human tumors is in codon 12 of theH-ras gene in which a base change from GGC to GTC results in aglycine-to-valine substitution in the GTPase regulatory domain of theras protein product. This single amino acid change is thought to abolishnormal control of ras protein function, thereby converting a normallyregulated cell protein to one that is continuously active. It isbelieved that such deregulation of normal ras protein function isresponsible for the transformation from normal to malignant growth.

While for illustrative purposes, the compounds of the invention aretargeted to ras RNA, it is of course recognized that a host of otherRNAs also are suitable as the target RNA. Thus the compounds of theinvention can be used to modulate the expression of any suitable targetRNA that is naturally present in cells or any target RNA in vitro.

The ras target site utilized for illustrative purposes is one the mostRNase H sensitive oligonucleotide sites that has been identified in theliterature. The selective inhibition of mutated genes such as the rasoncogene necessitates hybridization of a regulatory compound in thecoding region of the mRNA. This requires either a high affinityinteraction between such a compound and ras mRNA to prevent displacementof the compound by the polysome, or rapid degradation of the target mRNAby a given terminating mechanism. Again while not wishing to be bound bytheory, the RNA like compounds of the invention, have both inherentlyhigh affinity and are able to take advantage of the cellular dsRNaseactivity.

In accordance with the objects of this invention, novel oligomericcompounds that bind to a target RNA strand and that are substrates fordsRNase enzymes are provided. The oligomeric compounds of the inventioninclude oligoribonucleotides, oligoribonucleosides and other oligomericcompounds having a linear sequence of linked ribonucleoside subunitsincorporated therein. Such other oligomeric compounds will includechimeric structures formed between PNA (peptide nucleic acid) segmentsand linked ribonucleosides. Thus for the purposes of this specification,the term “oligomeric compound” is meant to be inclusive of the termsoligoribonucleotides and oligoribonucleosides, either used singly or incombination, as well as other oligomeric compounds including chimericcompounds formed between PNA segments (and other surrogate nucleosidecomponents) and linked ribonucleoside segments. As used in thisspecification and the claims attached hereto, in one sense the termoligomeric compound is used to represent oligoribonucleotides, in afurther sense to represent oligoribonucleosides, in even a further senseto represent mixtures of oligoribonucleotides and oligoribonucleosidesand in other instances to indicated further chimeric compounds such asthe above identified PNA chimeric compounds.

The oligoribonucleotides and oligoribonucleosides of the invention areassembled from a plurality of nucleoside subunits. In certain preferredoligoribonucleotide or oligoribonucleosides of the invention at leastone of the nucleoside subunits bear a substituent group that increasesthe binding affinity of the oligoribonucleotide or oligoribonucleosidefor a complementary strand of nucleic acid. Additionally, at least someof the nucleoside subunits comprise 2′-hydroxyl-pentofuranosyl sugarmoieties.

For cellular use, for an oligonucleotide to be particularly useful, theoligonucleotide must be reasonably stable to nucleases in order tosurvive in cells for a time period sufficient for it to interact withtarget nucleic acids of the cells. Therefore, in certain embodiments ofthe invention, specific nucleoside subunits or internucleoside linkagesare functionalized or selected to increase the nuclease resistance ofthe oligoribonucleotide or oligoribonucleoside. However, fornon-cellular uses, such as use of oligomeric compounds of the inventionas research reagents and as diagnostic agents, such nuclease stabilitymay not be necessary.

In determining the extent of binding affinity of a first nucleic acid toa complementary nucleic acid, the relative ability of the first nucleicacid to bind to the complementary nucleic acid may be compared bydetermining the melting temperature of a particular hybridizationcomplex. The melting temperature (T_(m)), a characteristic physicalproperty of double stranded nucleotides, denotes the temperature (indegrees centigrade) at which 50% helical (hybridized) versus coil(unhybridized) forms are present. T_(m) is measured by using the UVspectrum to determine the formation and breakdown (melting) of thehybridization complex. Base stacking which occurs during hybridizationis accompanied by a reduction in UV absorption (hypochromicity).Consequently, a reduction in UV absorption indicates a higher T_(m). Thehigher the T_(m), the greater the strength of the bonds between thestrands.

It has been found in the present invention that the binding affinity ofoligoribonucleotides and oligoribonucleosides of the present inventioncan be increased by incorporating substituent groups in the nucleosidesubunits of these compounds. Preferred substituent groups are 2′substituent groups, i.e. substituent groups located at the 2′ positionof the pentofuranosyl sugar moieties of the nucleoside subunits of thecompounds of the present invention. Presently preferred substituentgroups include fluoro, alkoxy, aminoalkoxy, allyloxy, imidazolylalkoxyand polyethylene glycol. Alkoxy and aminoalkoxy groups generally includelower alkyl groups, particularly C₁-C₉ alkyl. Polyethylene glycols areof the structure (O—CH₂—CH₂)_(n)—O-alkyl. A particularly preferredsubstituent group is a polyethylene glycol substituent of the formula(—O—CH₂—CH₂)—O-alkyl, wherein n=1 and alkyl=CH₃. This modification hasbeen shown to increase both affinity of a oligonucleotide for its targetand nuclease resistance of an oligonucleotide.

A further particularly useful 2′-substituent group for increasing thebinding affinity is the 2′-fluoro group. In a published study (Synthesisand Biophysical Studies of 2′-dRIBO-F Modified Oligonucleotides,Conference On Nucleic Acid Therapeutics, Clearwater, Fla., Jan. 13,1991) an increase in binding affinity of 1.6° C. per substitutednucleoside subunit was reported for a 15-mer phosphodiesteroligonucleotide having 2′-fluoro substituent groups on five of thenucleoside subunits of the oligonucleotide. When 11 of the nucleosidesubunits of the oligonucleotide bore 2′-fluoro substituent groups, thebinding affinity increased to 1.8° C. per substituted nucleosidesubunit. In this study, the 15-mer phosphodiester oligonucleotide wasderivatized to the corresponding phosphorothioate analog. When the15-mer phosphodiester oligonucleotide was compared to itsphosphorothioate analog, the phosphorothioate analog had a bindingaffinity of only about 66% of that of the 15-mer phosphodiesteroligonucleotide. Stated otherwise, binding affinity was lost inderivatizing the oligonucleotide to its phosphorothioate analog.However, when 2′-fluoro substituents were located on 11 of thenucleosides subunits of the 15-mer phosphorothioate oligonucleotide, thebinding affinity of the 2′-substituent groups more than overcame thedecrease noted by derivatizing the 15-mer oligonucleotide to itsphosphorothioate analog. In this compound, i.e. the 15-merphosphorothioate oligonucleotide having 11 nucleoside subunitssubstituted with 2′-fluoro substituent groups, the binding affinity wasincreased to 2.5° C. per substituent group.

For use in preparing the nucleoside structural subunits of the compoundsof the invention, suitable nucleobases for incorporation in thesenucleoside subunits include purines and pyrimidines such as adenine,guanine, cytosine, uridine, and thymine, as well as other synthetic andnatural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 5-halouracil andcytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine andthymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, amino, thiol,thioalkyl, hydroxyl and other 8-substituted adenines and guanines,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine. Further purines and pyrimidines include those disclosedin U.S. Pat. No. 3,687,808, those disclosed in the Concise EncyclopediaOf Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I.,ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613. Certain ofthese nucleobases are particularly useful for increasing the bindingaffinity of the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. Other modified pyrimidine and purine bases arealso expected to increase the binding affinity of oligomeric compoundsto a complementary strand of nucleic acid.

Preferred oligoribonucleotides and oligoribonucleosides in accordancewith this invention preferably comprise from about 5 to about 50nucleoside subunits. In the context of this invention it is understoodthat this encompasses non-naturally occurring oligomers as hereinbeforedescribed, having 5 to 50 nucleoside subunits. It is more preferred thatthe oligoribonucleotides and oligoribonucleosides of the presentinvention comprise from about 15 to about 25 nucleoside subunits. Aswill be appreciated, a “nucleoside subunit” is a nucleobase and sugar orsugar surrogate combination suitably bound to adjacent subunits throughphosphorus linkages in oligoribonucleotides and through non-phosphoruslinkages in oligoribonucleosides. In this context, the term “nucleosidesubunit” is used interchangeably with the term “nucleoside unit” or“nucleoside.”

The oligoribonucleotides of the invention have their nucleoside subunitsconnected by phosphorus linkages including phosphodiester,phosphorothioate, 3′-(or -5′)deoxy-3′-(or 5′)thio-phosphorothioate,phosphorodithioate, phosphoroselenates, 3′-(or -5′)deoxy phosphinates,borano phosphates, 3′-(or -5′)deoxy-3′-(or 5′-)amino phosphoramidates,hydrogen phosphonates, borano phosphate esters, phosphoramidates, alkylor aryl phosphonates and phosphotriester phosphorus linkages. Whereasthe oligoribonucleosides of the invention have their nucleoside subunitsconnected by carbonate, carbamate, silyl, sulfur, sulfonate,sulfonamide, formacetal, thioformacetyl, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino linkages.

In order to elicit a dsRNase response within the total overall sequencelength of the oligomeric compounds of the invention there will be asegment or subsequence of greater than three, but preferably, four, fiveor more consecutively linked 2′-hydroxyl-pentofuranosyl-containingnucleoside subunits. It is presently preferred to incorporate the2′-hydroxyl-pentofuranosyl-containing nucleoside subsequence in theoligomeric compound such that further subsequences or segments ofoligomeric compound are located on either side of the2′-hydroxyl-pentofuranosyl-containing nucleoside subsequence. In such aconstruction, the 2′-hydroxyl-pentofuranosyl containing nucleosidesubsequence is also referred to as the “central” or “gap” region orsegment and the other nucleoside subsequences or segments are referredto as “flanking” or “wing” regions or segments. Thus the “gap” region isflanked on either side by “wings.” Other constructions are alsopossible, including locating the 2′-hydroxylpentofuranosyl containingnucleoside subsequence at either the 3′ or the 5′ terminus of theoligomeric compound of the invention. These other constructions can beconsidered as “open” gapped structures, i.e., the gap region is open onthe end (either 3′ or 5′ end) of the oligomeric compound.

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be conveniently and routinely made through thewell-known technique of solid phase synthesis, see for example“Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRLPress, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed.F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aidedmethods of oligodeoxyribonucleotide synthesis, Chapter 2,Oligoribonucleotide synthesis, Chapter3,2′-O-Methyloligoribonucleotides: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein.

Equipment for oligonucleotide and oligonucleoside synthesis is sold byseveral vendors including Applied Biosystems. Various amidite reagentsare also commercially available, including 2′O-methyl amidites and2′-O-hydroxylamidites. Any other means for such synthesis may also beemployed. The actual synthesis of the oligonucleotides is well withinthe talents of those skilled in the art. It is also well known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives. It is also well known touse similar techniques and commercially available modified amidites andcontrolled-pore glass (CPG) products such as biotin, fluorescein,acridine or psoralen-modified amidites and/or CPG (available from GlenResearch, Sterling Va.) to synthesize fluorescently labeled,biotinylated or other conjugated oligonucleotides.

In a further embodiment, the present invention is drawn to a mammalianribonuclease isolatable from human T24 cells, and other cell lines, thatdegrades RNA in an antisense oligoribonucleotide:RNA duplex. Theribonuclease is referred to herein as a dsRNase, wherein “ds” indicatesthe RNase's specificity for double-stranded RNA substrates. Antisenseoligodeoxynucleotides containing 2′-methoxy modified sugar moieties bindto their cellular mRNA targets with high affinity but the resulting[“DNA-like”]:[RNA] duplexes are not substrates for nucleolyticdegradation in T24 cells. As detailed in the Examples, 2′-methoxyphosphorothioate antisense oligonucleotides targeting codon 12 of Ha-RasmRNA were modified by substituting 2′-methoxy nucleotides with a stretchof ribonucleotides in the center of the oligonucleotide to form2′-methoxy/ribo/2′-methoxy chimeric or “gapmer” oligonucleotides, withthe phosphorothioate linkage maintained throughout the molecules. These“RNA-like” gapmer oligonucleotides bind to their cellular mRNA targetwith an affinity comparable to that of the full 2′-methoxyoligodeoxynucleotide, but, unlike the [“DNA-like”]:[RNA] duplexes, theresultant [“RNA-like”]:[RNA] duplexes are substrates for nucleolyticdegradation in T24 cells. Degradation of the [antisense “RNA-like”gapmer oligonucleotide]:[Ha-Ras mRNA] duplex is dependent on the numberof ribonucleotides incorporated into the antisense molecule. A 17 basepair 9 RNA gapmer oligonucleotide:RNA duplex is not a substrate forRNase H cleavage, but is a substrate for cleavage by an the dsRNase ofthe invention in T24 cellular lysates. Furthermore, the cleavage sitesseen with T24 cellular lysates are localized to the RNA:RNA portion ofthe duplex and are not seen in the 2′-methoxy:RNA portion of the duplex.Cleavage of the duplex by the dsRNase of the invention produces5″-phosphate and 3″-hydroxyl termini.

Compounds of the invention can be utilized as diagnostics, therapeuticsand as research reagents and kits. They can be utilized inpharmaceutical compositions by adding an effective amount of a compoundof the invention to a suitable pharmaceutically acceptable diluent orcarrier. They further can be used for treating organisms having adisease characterized by the undesired production of a protein. Theorganism can be contacted with a compound of the invention having asequence that is capable of specifically hybridizing with a strand oftarget nucleic acid that codes for the undesirable protein.

The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.In general, for therapeutics, a patient in need of such therapy isadministered a compound in accordance with the invention, commonly in apharmaceutically acceptable carrier, in doses ranging from 0.01 μg to100 g per kg of body weight depending on the age of the patient and theseverity of the disease state being treated. Further, the treatmentregimen may last for a period of time which will vary depending upon thenature of the particular disease, its severity and the overall conditionof the patient, and may extend from once daily to once every 20 years.Following treatment, the patient is monitored for changes in his/hercondition and for alleviation of the symptoms of the disease state. Thedosage of the compound may either be increased in the event the patientdoes not respond significantly to current dosage levels, or the dose maybe decreased if an alleviation of the symptoms of the disease state isobserved, or if the disease state has been ablated.

In some cases it may be more effective to treat a patient with acompound of the invention in conjunction with other traditionaltherapeutic modalities. For example, a patient being treated for a viraldisease may be administered a compound of the invention in conjunctionwith a known antiviral agent, or a patient with atherosclerosis may betreated with a compound of the invention following angioplasty toprevent reocclusion of the treated arteries.

Following successful treatment, it may be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the compound of the invention is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water or non-aqueous media, capsules,sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives.

Dosing is dependent on severity and responsiveness of the diseasecondition to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of disease state is achieved. Optimal dosing schedules can becalculated from measurements of drug accumulation in the body of thepatient. Persons of ordinary skill can easily determine optimum dosages,dosing methodologies and repetition rates. Optimum dosages may varydepending on the relative potency of individual compounds, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years.

Such treatment can be practiced in a variety of organisms ranging fromunicellular prokaryotic and eukaryotic organisms to multicellulareukaryotic organisms. Any organism that utilizes DNA-RNA transcriptionor RNA-protein translation as a fundamental part of its hereditary,metabolic or cellular machinery is susceptible to such diagnostic,therapeutic and/or prophylactic treatment. Seemingly diverse organismssuch as bacteria, yeast, protozoa, algae, plant and higher animal forms,including warm-blooded animals, can be treated in this manner. Further,since each of the cells of multicellular eukaryotes also includes bothDNA-RNA transcription and RNA-protein translation as an integral part oftheir cellular activity, such therapeutics and/or diagnostics can alsobe practiced on such cellular populations. Furthermore, many of theorganelles, e.g. mitochondria and chloroplasts, of eukaryotic cells alsoinclude transcription and translation mechanisms. As such, single cells,cellular populations or organelles also can be included within thedefinition of organisms that are capable of being treated with thetherapeutic or diagnostic compounds of the invention. As used herein,therapeutics is meant to include eradication of a disease state, killingof an organism, e.g. bacterial, protozoan or other infection, or controlof aberrant or undesirable cellular growth or expression.

In the context of this invention, “target RNA” shall mean any RNA thatcan hybridize with a complementary nucleic acid like compound. Furtherin the context of this invention, “hybridization” shall mean hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleobases. “Complementary” asused herein, refers to the capacity for precise pairing between twonucleobases. For example, adenine and thymine are complementarynucleobases which pair through the formation of hydrogen bonds.“Complementary” and “specifically hybridizable,” as used herein, referto precise pairing or sequence complementarity between a first and asecond nucleic acid-like oligomers containing nucleoside subunits. Forexample, if a nucleobase at a certain position of the first nucleic acidis capable of hydrogen bonding with a nucleobase at the same position ofthe second nucleic acid, then the first nucleic acid and the secondnucleic acid are considered to be complementary to each other at thatposition. The first and second nucleic acids are complementary to eachother when a sufficient number of corresponding positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity suchthat stable and specific binding occurs between a compound of theinvention and a target RNA molecule. It is understood that an oligomericcompound of the invention need not be 100% complementary to its targetRNA sequence to be specifically hybridizable. An oligomeric compound isspecifically hybridizable when binding of the oligomeric compound to thetarget RNA molecule interferes with the normal function of the targetRNA to cause a loss of utility, and there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligomeric compoundto non-target sequences under conditions in which specific binding isdesired, i.e. under physiological conditions in the case of in vivoassays or therapeutic treatment, or in the case of in vitro assays,under conditions in which the assays are performed.

The following examples and procedures illustrate the present inventionand are not intended to limit the same. In illustrating the invention,Example 1 identifies certain commercial nucleoside amidites and otheradditional nucleoside amidites that are useful for the preparation ofcertain illustrative oligoribonucleotide or oligoribonucleosidecompounds of the invention. Examples 2 through 5 illustrate thepreparation of further nucleoside amidites use in preparing otherillustrative oligoribonucleotide or oligoribonucleoside compounds of theinvention. Example 6 illustrates the preparation of oligoribonucleotidecompounds of the invention. Example 7 illustrates the preparation ofoligoribonucleoside compounds of the invention. Examples 8 through 16illustrate the preparation of chimeric oligomeric compounds of theinvention including certain “gapmers,” i.e., compounds having “gap” and“wing” constructions. Examples 17 through 18 illustrate certain usefulaspects of the compounds of the invention. Examples 19 through 28illustrate the identification, characterization and purification of thedouble-stranded ribonucleases (dsRNases) of the invention. Example 29illustrates affinity columns incorporating the dsRNase substrates of theinvention.

In the illustrative examples, several different types of “gapmers” areexemplified. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. In the illustrative examples, for all chimericoligoribonucleotides and oligoribonucleosides, unless otherwiseindicated, 2′-O-methyl nucleosides are utilized in the “wing” segmentsand 2′-OH nucleosides are utilized in the “gap” segments of therespective oligoribonucleotides or oligoribonucleosides.

For the purposes of the illustrative examples the following short handconventions are used. Structure set forth in brackets, i.e. [ ], arenucleoside abbreviations, while structures set forth following a slashmark, i.e. /, are linkers used to connect the nucleosides, i.e. backbonestructures that link the nucleosides together in eitheroligoribonucleotide or oligoribonucleoside compounds.

Using this nomenclature, the following abbreviations are used forphosphate linkages between nucleosides: PO for phosphodiester; PS forphosphorothioate, P2S for phosphorodithioate, PSe forphosphoroselenates, PMe for methyl phosphonate, POMe for methylphosphotriester, PN for phosphoramidate, 3′NPN for 3′-deoxy-3′-aminophosphoramidate, PI for phosphinate, MePS for alkylphosphonothioate, BPfor borano phosphate are used. For non-phosphate linkages betweennucleosides the abbreviations used are: MMI for methylenemethylimino,MDH for methylenedimethylhydrazo, FA for formacetal, TFA forthioformacetal, ETO for ethylene oxide and amide-3 formethylenecarbonylamino. 2′-OH is utilized as an abbreviation forunmodified ribo sugars, i.e. pentoribofuranosyl sugars. For modifiednucleosides the abbreviations used are: 2′-O-alkyl for general alkylgroups at the 2′ position of a pentoribofuranosyl moiety with specificalkyl being noted as 2′-O-Me, 2′-O-Et, 2′-O-Pr and 2′-O-EtOMe formethyl, ethyl, propyl and methoxyethyl, respectively; 2′-F for a fluoromoiety at the 2′ position of a pentoribofuranosyl moiety, Mod-Purine fora purine nucleobase substitution as, for example, per the disclosure ofU.S. Pat. No. 5,459,255 or; and Mod-Pyr for a pyrimidine nucleobasesubstitution as, for example, per the disclosure of U.S. Pat. No.5,484,908; SS for a sugar surrogate as, for example, per the disclosureof U.S. Pat. No. 5,359,044.

Example 1 Amidites for Oligonucleotide/Oligonucleoside Synthesis

2′-O-Methyl nucleoside amidites and 2′-OH (blocked as2′-t-butyldimethylsilyl derivative) nucleoside amidites are availablefrom Glen Research, Sterling, Va. Other 2′-O-alkyl substitutednucleoside amidites are prepared as is described in U.S. Pat. No.5,506,351, 5,466,786 or 5,514,786, herein incorporated by reference.Cyclobutyl sugar surrogate compounds are prepared as is described inU.S. Pat. No. 5,359,044, herein incorporated by reference. Pyrrolidinesugar surrogate are prepared as is described in U.S. Pat. No. 5,519,134,herein incorporated by reference. Morpholino sugar surrogates areprepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,herein incorporated by reference, and other related patent disclosures.N-2 substituted purine nucleoside amidites are prepared as is describedin U.S. Pat. No. 5,459,255, herein incorporated by reference. 3-Deazapurine nucleoside amidites are prepared as is described in U.S. Pat. No.5,457,191, herein incorporated by reference. 5,6-Substituted pyrimidinenucleoside amidites are prepared as is described in U.S. Pat. No.5,614,617 herein incorporated by reference. 5-Propynyl pyrimidinenucleoside amidites are prepared as is described in U.S. Pat. No.5,484,908, herein incorporated by reference.

Example 2 2′-O-(Methoxyethyl) Nucleoside Amidites

2′-O-Ethyl-O-methyl substituted nucleoside amidites are prepared asfollows in Examples 2-a through 2-h or alternately, as per the methodsof Martin, P., Helvetica Chimica Acta, 1995, 78, 486-504.

Example 2-a 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]

5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60° C. at 1 mm Hg for24 h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The NMR spectrum was consistent with the structure,contaminated with phenol as its sodium salt (ca. 5%). The material wasused as is for further reactions (or it can be purified further bycolumn chromatography using a gradient of methanol in ethyl acetate(10-25%) to give a white solid, mp 222-4° C.).

Example 2-b 2′-O-Methoxyethyl-5-methyluridine

2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate(231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 Lstainless steel pressure vessel and placed in a pre-heated oil bath at160° C. After heating for 48 hours at 155-160° C., the vessel was openedand the solution evaporated to dryness and triturated with MeOH (200mL). The residue was suspended in hot acetone (1 L). The insoluble saltswere filtered, washed with acetone (150 mL) and the filtrate evaporated.The residue (280 g) was dissolved in CH₃CN (600 mL) and evaporated. Asilica gel column (3 kg) was packed in CH₂Cl₂/acetone/MeOH (20:5:3)containing 0.5% Et₃NH. The residue was dissolved in CH₂Cl₂ (250 mL) andadsorbed onto silica (150 g) prior to loading onto the column. Theproduct was eluted with the packing solvent to give 160 g (63%) ofproduct. Additional material was obtained by reworking impure fractions.

Example 2-c 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporatedwith pyridine (250 mL) and the dried residue dissolved in pyridine (1.3L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) wasadded and the mixture stirred at room temperature for one hour. A secondaliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and thereaction stirred for an additional one hour. Methanol (170 mL) was thenadded to stop the reaction. HPLC showed the presence of approximately70% product. The solvent was evaporated and triturated with CH₃CN (200mL). The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500mL of saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).

Example 2-d3′-O-Acetyl-2′-O-methoxyethyl-5-O-dimethoxytrityl-5-methyluridine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35° C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporate to give 122 g of residue (approx. 90%product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%). An additional 1.5 g was recovered from laterfractions.

Example 2-e3′-O-Acetyl-2′-O-methoxyethyl-5-O-dimethoxytrityl-5-methyl-4-triazoleuridine

A first solution was prepared by dissolving3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96g, 0.144 M) in CH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L),cooled to −5° C. and stirred for 0.5 h using an overhead stirrer. POCl₃was added dropwise, over a 30 minute period, to the stirred solutionmaintained at 0-10° C., and the resulting mixture stirred for anadditional 2 hours. The first solution was added dropwise, over a 45minute period, to the later solution. The resulting reaction mixture wasstored overnight in a cold room. Salts were filtered from the reactionmixture and the solution was evaporated. The residue was dissolved inEtOAc (1 L) and the insoluble solids were removed by filtration. Thefiltrate was washed with 1×300 mL of NaHCO₃ and 2×300 mL of saturatedNaCl, dried over sodium sulfate and evaporated. The residue wastriturated with EtOAc to give the title compound.

Example 2-f 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

A solution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. MeOH (400 mL) saturated with NH₃ gas was added and the vesselheated to 100° C. for 2 hours (tic showed complete conversion). Thevessel contents were evaporated to dryness and the residue was dissolvedin EtOAc (500 mL) and washed once with saturated NaCl (200 mL). Theorganics were dried over sodium sulfate and the solvent was evaporatedto give 85 g (95%) of the title compound.

Example 2-gN⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine

2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.

Example 2-hN⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite

N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tic showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1×300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound.

Example 3 Preparation of Long Chain, i.e. (C₂₀), Substituted NucleosideAmidites

Synthesis of nucleoside amidites having long chains, e.g. C₂₀,substituents at their 2′ position is shown in Examples 3-a through 3-c.

Example 3-a Synthesis of2,6-Diamino-9-(2-O-octadecyl-β-D-ribofuranosyl)purine

2,6-Diamino-9-(β-D-ribofuranosyl)purine (50 g, 180 mmol) and sodiumhydride (7 g) in DMF (1 L) were heated to boiling for 2 hr.Iodooctadecane (100 g) was added at 150° C. and the reaction mixtureallowed to cool to RT. The reaction mixture was stirred for 11 days atRT. The solvent was evaporated and the residue purified by silica gelchromatography. The product was eluted with 5% MeOH/CH₂Cl₂. Theappropriate fractions were evaporated to yield the product (11 g). ¹HNMR (DMSO-d₆) δ 0.84 (t, 3, CH₂); 1.22 (m, 32, O—CH₂—CH₂—(CH₂)₁₆); 1.86(m, 2, O—CH₂CH₂); 3.25 (m, 2, O—CH₂); 3.93 (d, 1, 4′H), 4.25 (m, 1,3′H); 4.38 (t, 1, 2′H); 5.08 (d, 1,3′-OH); 5.48 (t, 1,5′-OH); 5.75 (s,2, 6-NH₂); 5.84 (d, 1, 1′-H); 6.8 (s, 2, 2-NH₂); and 7.95 (s, 1, 8-H).

Example 3-b Synthesis of 2′-O-Octadecylguanosine

2,6-Diamino-9-(2-O-octadecyl-β-D-ribofuranosyl) purine (10 g) in 0.1 Msodium phosphate buffer (50 mL, pH 7.4), 0.1 M tris buffer (1000 mL, pH7.4) and DMSO (1000 mL) was treated with adenosine deaminase (1.5 g) atRT. At day 3, day 5 and day 7 an additional aliquot (500 mg, 880 mg and200 mg, respectively) of adenosine deaminase was added. The reaction wasstirred for a total of 9 day and purification by silica gelchromatography yielded the product (2 g). An analytical sample wasrecrystallized from MeOH ¹H NMR (DMSO-d₆) δ 0.84 (t, 3, CH₃), 1.22 [s,32, O—CH₂—CH₂—(CH₂)₁₆], 5.07 (m, 2,3′-OH and 5′-OH); 5.78 (d, 1, 1′-H);6.43 (s, 2, NH₂), 7.97 (s, 1, 8-H) and 10.64 (s, 1, NH₂). Anal. Calcd.for C₂₈H₄₉N₅O₅: C, 62.80; H, 9.16; N, 12.95. Found: C, 62.54; H, 9.18;N, 12.95.

Example 3-c Synthesis of N²-Isobutyryl-2′-O-octadecylguanosine

2′-O-Octadecylguanosine (1.9 g) in pyridine (150 mL) was cooled in anice bath, and treated with trimethylsilyl chloride (2 g, 5 eq) andisobutyryl chloride (2 g, 5 eq). The reaction mixture was stirred for 4hours, during which time it was allowed to warm to room temperature. Thesolution was cooled, water added (10 mL) and stirred for an additional30 minutes. Concentrated ammonium hydroxide (10 mL) was added and thesolution concentrated in vacuo. The residue was purified by silica gelchromatography (eluted with 3% MeOH/EtOAc) to yield 1.2 g of product. ¹HNMR (DMSO-d₆) δ 0.85 (t, 3, CH₃), 1.15 (m, 38, O—CH₂CH₂(CH₂)₁₆,CH(CH₃)₂), 2.77 (m, 1, CH(CH₃)₂), 4.25 (m, 2,2′-H and 3′-H); 5.08 (t,1,5′-OH), 5.12 (d, 1,3′-OH), 5.87 (d, 1,1′-H), 8.27 (s, 1,8-H), 11.68(s, 1, NH₂) and 12.08 (s, 1, NH₂). Anal. Calcd. for C₃₂H₅₅N₅O₆: C,63.47; H, 9.09; N, 11.57. Found: C, 63.53; H, 9.20; N, 11.52. Prior toincorporating this product into an oligonucleotide, it was converted toN²-Isobutyryl-5′-dimethoxytrityl-2′-O-octadecylguanosine and then to aphosphoramidite according to the procedures described in InternationalPublication Number WO 94/02501, published Feb. 3, 1994.

Example 4 2′-Fluoro Nucleoside Amidites

2′-fluoro substituted nucleoside amidites are prepared as follows inExamples 4-a through 4-d or alternately as per the method of Kawasakiet. al., J. Med. Chem., 1993, 36, 831-841.

Example 4-a i. N⁶-Benzoyl-9-β-D-arabinofuranosyladenine

9-β-D-arabinofuranosyladenine (1.07 g, 4.00 mmol) was dissolved inanhydrous pyridine (20 mL) and anhydrous dimethylformamide (20 mL) underan argon atmosphere. The solution was cooled to 0° C. andchlorotrimethylsilane (3.88 mL, 30.6 mmol) was added slowly to thereaction mixture via a syringe. After stirring the reaction mixture at0° C. for 30 minutes, benzoyl chloride (2.32 mL, 20 mmol) was addedslowly. The reaction mixture was allowed to warm to 20° C. and stirredfor 2 hours. After cooling the reaction mixture to 0° C., cold water (8mL) was added and the mixture was stirred for 15 minutes. Concentratedammonium hydroxide (8 mL) was slowly added to the reaction mixture togive a final concentration of 2 M of ammonia. After stirring the coldreaction mixture for 30 minutes, the solvent was evaporated in vacuo (60torr) at 20° C. followed by evaporation in vacuo (1 torr) at 40° C. togive an oil. This oil was triturated with diethyl ether (50 mL) to givea solid which was filtered and washed with diethyl ether three times.This crude solid was triturated in methanol (100 mL) at refluxtemperature three times and the solvent was evaporated to yieldN⁶-Benzoyl-9-β-D-arabino-furanosyladenine as a solid (1.50 g, 100%).

ii. N⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl] adenine

N⁶-Benzoyl-9-β-D-arabinofuranosyladenine (2.62 g, 7.06 mmol) wasdissolved in anhydrous dimethylformamide (150 mL) under argon andp-toluenesulfonic acid monohydrate (1.32 g, 6.92 mmol) was added. Thissolution was cooled to 0° C. and dihydropyran (1.26 mL, 13.8 mmol) wasadded via a syringe. The reaction mixture was allowed to warm to 20° C.Over a period of 5 hours a total of 10 equivalents of dihydropyran wereadded in 2 equivalent amounts in the fashion described. The reactionmixture was cooled to 0° C. and saturated aqueous sodium bicarbonate wasadded slowly to a pH of 8, then water was added to a volume of 750 mL.The aqueous mixture was extracted with methylene chloride (4×200 mL),and the organic phases were combined and dried over magnesium sulfate.The solids were filtered and the solvent was evaporated in vacuo (60ton) at 30° C. to give a small volume of liquid which was evaporated invacuo (1 torr) at 40° C. to give an oil. This oil was coevaporated withp-xylene in vacuo at 40° C. to give an oil which was dissolved inmethylene chloride (100 mL). Hexane (200 mL) was added to the solutionand the lower-boiling solvent was evaporated in vacuo at 30° C. to leavea white solid suspended in hexane. This solid was filtered and washedwith hexane (3×10 mL) then purified by column chromatography usingsilica gel and methylene chloride-methanol (93:7) as the eluent. Thefirst fraction yielded the title compound 3 as a white foam (3.19 g,83%) and a second fraction gave a white foam (0.81 g) which wascharacterized as the 5′-monotetrahydropyranyl derivative ofN⁶-Benzoyl-9-β-D-arabinofuranosyladenine.

iii.N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine

N⁶-Benzoyl-9-[3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(2.65 g, 4.91 mmol) was dissolved in anhydrous pyridine (20 mL) and thesolvent was evaporated in vacuo (1 mm Hg) at 40° C. The resulting oilwas dissolved in anhydrous methylene chloride (130 mL) under argonanhydrous pyridine (3.34 mL, 41.3 mmol) and N,N-dimethylaminopyridine(1.95 g, 16.0 mmol) were added. The reaction mixture was cooled to 0° C.and trifluoromethanesulfonic anhydride (1.36 mL, 8.05 mmol) was addedslowly via a syringe. After stirring the reaction mixture at 0° C. for 1hour, it was poured into cold saturated aqueous sodium bicarbonate (140mL). The mixture was shaken and the organic phase was separated and keptat 0° C. The aqueous phase was extracted with methylene chloride (2×140mL). The organic extracts which were diligently kept cold were combinedand dried over magnesium sulfate. The solvent was evaporated in vacuo(60 ton) at 20° C. then evaporated in vacuo (1 torr) at 20° C. to giveN⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenineas a crude oil which was not purified further.

iv.N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine

N⁶-Benzoyl-9-[2′-O-trifluoromethylsulfonyl-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(4.9 mmol) as a crude oil was dissolved in anhydrous tetrahydrofuran(120 mL) and this solution was cooled to 0° C. under argon.Tetrabutylammonium fluoride as the hydrate (12.8 g, 49.1 mmol) wasdissolved in anhydrous tetrahydrofuran (50 mL) and half of this volumewas slowly added via a syringe to the cold reaction mixture. Afterstirring at 0° C. for 1 hour, the remainder of the reagent was addedslowly. The reaction mixture was stirred at 0° C. for an additional 1hour, then the solvent was evaporated in vacuo (60 ton) at 20° C. togive an oil. This oil was dissolved in methylene chloride (250 mL) andwashed with brine three times. The organic phase was separated and driedover magnesium sulfate. The solids were filtered and the solvent wasevaporated to give an oil. The crude product was purified by columnchromatography using silica gel in a sintered-glass funnel and ethylacetate was used as the eluent. N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O—tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine was obtained as anoil (2.03 g, 76%).

v. N⁶-Benzoyl-9-(2′-fluoro-β-D-ribofuranosyl)adenine

N⁶-Benzoyl-9-[2′-fluoro-3′,5′-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl]adenine(1.31 g, 2.42 mmol) was dissolved in methanol (60 mL), and Dowex50W×2-100 (4 cm³, 2.4 m.eq) was added to the reaction mixture. Thereaction mixture was stirred at 20° C. for 1 hour then cooled to 0° C.Triethylamine (5 mL) was then slowly added to the cold reaction mixtureto a pH of 12. The resin was filtered and washed with 30% triethylaminein methanol until the wash no longer contained UV absorbing material.Toluene (50 mL) was added to the washes and the solvent was evaporatedat 24° C. in vacuo (60 ton, then 1 ton) to give a residue. This residuewas partially dissolved in methylene chloride (30 mL) and the solventwas transferred to a separatory funnel. The remainder of the residue wasdissolved in hot (60° C.) water and after cooling the solvent it wasalso added to the separatory funnel. The biphasic system was extracted,and the organic phase was separated and extracted with water (3×100 mL).The combined aqueous extracts were evaporated in vacuo (60 ton, then 1torr Hg) at 40° C. to give an oil which was evaporated with anhydrouspyridine (50 mL). This oil was further dried in vacuo (1 ton Hg) at 20°C. in the presence of phosphorous pentoxide overnight to giveN⁶-benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine as a yellow foam (1.08g, 100%) which contained minor impurities.

vi.N⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4-dimethoxy-trityl)-β-D-ribofuranosyl]adenine

N⁶-Benzoyl-9-(2′-fluoro-b-D-ribofuranosyl)adenine (1.08 g, 2.89 mmol)which contained minor impurities was dissolved in anhydrous pyridine (20mL) under argon and dry triethylamine (0.52 mL, 3.76 mmol) was addedfollowed by addition of 4,4′-dimethoxytrityl chloride (1.13 g, 3.32mmol). After 4 hours of stirring at 20° C. the reaction mixture wastransferred to a separatory funnel and diethyl ether (40 mL) was addedto give a white suspension. This mixture was washed with water threetimes (3×10 mL), the organic phase was separated and dried overmagnesium sulfate. Triethylamine (1 mL) was added to the solution andthe solvent was evaporated in vacuo (60 torr Hg) at 20° C. to give anoil which was evaporated with toluene (20 mL) containing triethylamine(1 mL). This crude product was purified by column chromatography usingsilica gel and ethyl acetate-triethylamine (99:1) followed by ethylacetate-methanol-triethylamine (80:19:1) to give the product in twofractions. The fractions were evaporated in vacuo (60 ton, then 1 torrHg) at 20° C. to give a foam which was further dried in vacuo (1 ton Hg)at 20° C. in the presence of sodium hydroxide to giveN⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]adenineas a foam (1.02 g, 52%).

vii. N⁶-Benzoyl-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)]-adenosine-3′-O—N,N-diisopropyl-β-cyanoethyl phosphoramidite

N⁶-Benzoyl-9-[2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-β-D-ribofuranosyl]adenine(1.26 g, 1.89 mmol) was dissolved in anhydrous dichloromethane (13 mL)under argon, diisopropylethylamine (0.82 mL, 4.66 mmol) was added, andthe reaction mixture was cooled to 0° C.Chloro(diisopropylamino)-O-cyanoethoxyphosphine (0.88 mL, 4.03 mmol) wasadded to the reaction mixture which was allowed to warm to 20° C. andstirred for 3 hours. Ethylacetate (80 mL) and triethylamine (1 mL) wereadded and this solution was washed with brine (3×25 mL). The organicphase was separated and dried over magnesium sulfate. After filtrationof the solids the solvent was evaporated in vacuo at 20° C. to give anoil which was purified by column chromatography using silica gel andhexanes-ethyl acetate-triethyl-amine (50:49:1) as the eluent.Evaporation of the fractions in vacuo at 20° C. gave a foam which wasevaporated with anhydrous pyridine (20 mL) in vacuo (1 ton) at 26° C.and further dried in vacuo (1 torr Hg) at 20° C. in the presence ofsodium hydroxide for 24 h to giveN⁶-Benzoyl-[T-deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)]-adenosine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramiditeas a foam (1.05 g, 63%).

Example 4-b2′-Deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-uridine-3′O(N,N-diisopropyl-β-cyanoethyl-phosphoramidite)

2,2′-Cyclouridine is treated with a solution of 70% hydrogenfluoride/pyridine in dioxane at 120° C. for ten hours to provide aftersolvent removal a 75% yield of 2′-deoxy-2′-fluorouridine. The 5′-DMT and3′-cyanoethoxydiisopropyl-phosphoramidite derivitized nucleoside isobtained by standard literature procedures [Gait, Ed., OligonucleotideSynthesis. A Practical Approach, IRL Press, Washington, D.C. (1984)], oraccording to the procedure of Example 4-a.

Example 4-c2′-Deoxy-2′-fluoro-5′-O-(4,4′-dimethoxytrityl)-cytidine-3′-O—(N,N-diisopropyl-β-cyanoethylphosphoramidite)

2′-Deoxy-2′-fluorouridine (2.51 g, 10.3 mmol) was converted tocorresponding cytidine analog via the method of C. B. Reese, et al., J.Chem. Soc. Perkin Trans I, pp. 1171-1176 (1982), by acetylation withacetic anhydride (3.1 mL, 32.7 mmol) in anhydrous pyridine (26 mL) atroom temperature. The reaction was quenched with methanol, the solventwas evaporated in vacuo (1 ton) to give an oil which was coevaporatedwith ethanol and toluene. 3′,5′-O— diacetyl-2′-deoxy-2′-fluorouridinewas crystallized from ethanol to afford colorless crystals (2.38 g,81%).

N-4-(1,2,4-triazol-1-yl)-3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wasobtained in a 70% yield (2.37 g) by reaction of3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine (2.75 g, 9.61 mmol) with1,2,4-triazole (5.97 g, 86.5 mmol), phosphorus oxychloride (1.73 mL,18.4 mmol), and triethylamine (11.5 mL, 82.7 mmol) in anhydrousacetonitrile at room temperature. After 90 min the reaction mixture wascooled to ice temperature and triethylamine (7.98 ml, 56.9 mmol) wasadded followed by addition of water (4.0 ml). The solvent was evaporatedin vacuo (1 torr) to give an oil which was dissolved in methylenechloride and washed with saturated aqueous sodium bicarbonate. Theaqueous phase was extracted with methylene chloride twice (2×100 mL) andthe organic extracts dried with magnesium sulfate. Evaporation of thesolvent afforded an oil from which the productN-4-(1,2,4-triazol-1-yl)-3′,5′-O-diacetyl-2′-deoxy-2′-fluorouridine wasobtained by crystallization from ethanol.

2′-deoxy-2′-fluorocytidine was afforded by treatment of protectedtriazol-1-yl derivative with concentrated ammonium hydroxide (4.26 mL,81.2 mmol) in dioxane at room temperature for 6 hours. After evaporationof the solvent the oil was stirred in half-saturated (at icetemperature) ammonia in methanol for 16 hours. The solvent wasevaporated and 2′-deoxy-2′-fluoro-cytidine crystallized fromethylacetate-methanol (v/v, 75:25) to give colorless crystals (1.24 g,75%).

N-4-benzoyl-2′-deoxy-2′-fluorocytidine was prepared by selectivebenzoylation with benzoic anhydride in anhydrous dimethylformamide, V.Bhat, et al. Nucleosides Nucleotides, Vol. 8, pp. 179-183 (1989). The5′-O-(4,4′-dimethoxytrityl)-3′-O—(N,N-diisopropyl-β-cyanoethyl-phosphoramidite)was prepared in accordance with Example 4-a.

Example 4-d i.9-(3′,5′-[1,1,3,3-Tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine

The 3′ and 5′ positions of guanosine were protected by the addition of aTPDS (1,1,3,3-tetraisopropyldisilox-1,3-diyl) protecting group as perthe procedure of Robins et al. [Can. J. Chem., 61, 1911 (1983)]. To astirred solution of DMSO (160 mL) and acetic anhydride (20 mL) was addedthe TPDS guanosine (21 g, 0.040 mol). The reaction was stirred at roomtemperature for 36 hours and then cooled to 0° C. Cold ethanol (400 mL,95%) was added and the reaction mixture further cooled to −78° C. in adry ice/acetone bath. NaBH₄ (2.0 g, 1.32 mol. eq.) was added. Thereaction mixture was allowed to warm up to −2° C., stirred for 30minutes and again cooled to −78° C. This was repeated twice. After theaddition of NaBH₄ was complete, the reaction was stirred at 0° C. for 30minutes and then at room temperature for 1 hour. The reaction was takenup in ethyl acetate (1 L) and washed twice with a saturated solution ofNaCl. The organic layer was dried over MgSO₄ and evaporated underreduced pressure. The residue was coevaporated twice with toluene andpurified by silica gel chromatography using CH₂Cl₂-MeOH (9:1) as theeluent. Pure product (6.02 g) precipitated from the appropriate columnfractions during evaporation of these fractions, and an additional 11.49g of product was obtained as a residue upon evaporation of thefractions.

ii.N²-Isobutyryl-9-(2′-O-isobutyryl-3′,5′-[1,1,3,3-tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine

9-(3′,5′-[1,1,3,3-Tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine(6.5 g, 0.01248 mol) was dissolved in anhydrous pyridine (156 mL) underargon. DMAP (9.15 g) was added. Isobutyric anhydride (6.12 mL) wasslowly added and the reaction mixture stirred at room temperatureovernight. The reaction mixture was poured into cold saturated NaHCO₃(156 mL) and stirred for 10 minutes. The aqueous solution was extractedthree times with ethyl acetate (156 mL). The organic phase was washedthree times with saturated NaHCO₃ and evaporated to dryness. The residuewas coevaporated with toluene and purified by silica gel columnchromatography using CH₂Cl₂-acetone (85:15) to yield 5.67 g of product.

iii. N²-Isobutyryl-9-(2′-O-isobutyryl-β-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(2′-isobutyryl-3′,5′-[1,1,3,3-tetraisopropyldisilox-1,3-diyl]-β-D-arabinofuranosyl)guanine(9.83 g, 0.01476 mol) was dissolved in anhydrous THF (87.4 mL) at roomtemperature under argon. 1 M (nBu)₄N⁺F⁻ in THF (29.52 mL, 2 eq.) wasadded and the reaction mixture stirred for 30 minutes. The reactionmixture was evaporated at room temperature and the residue purified bysilica gel column chromatography using EtOAc-MeOH (85:15) to yield 4.98g (80%) of product.

iv.N²-Isobutyryl-9-(2′-O-isobutyryl-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(2′-isobutyryl-β-D-arabinofuranosyl)guanine (4.9 g) wasdissolved in anhydrous 1,4-dioxane (98 mL) at room temperature underargon. p-Toluenesulphonic acid monohydrate (0.97 g) was added followedby 3,4-dihydro-2H-pyran (DHP, 9.34 mL, 8.8 eq.). The reaction mixturewas stirred for 2 hours, then cooled to 0° C. and saturated NaHCO₃ (125mL) was added to quench the reaction. The reaction mixture was extractedthree times with 125 mL portions of CH₂Cl₂ and the organic phase driedover MgSO₄. The organic phase was evaporated and the residue dissolvedin minimum volume of CH₂Cl₂, but in an amount sufficient to yield aclear liquid not a syrup, and then dripped into hexane (100 times thevolume of CH₂Cl₂). The precipitate was filtered to yield 5.59 (81.5%) ofproduct.

v.N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(2′-isobutyryl-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine(5.58 g) was dissolved in pyridine-MeOH—H₂O (65:30:15, 52 mL) at roomtemperature. The solution was cooled to 0° C. and 52 mL of 2 N NaOH inEtOH-MeOH (95:5) was added slowly, followed by stirring for 2 hours at0° C. Glacial acetic acid was added to pH 6, and saturated NaHCO₃ wasadded to pH 7. The reaction mixture was evaporated under reducedpressure and the residue coevaporated with toluene. The residue was thendissolved in EtOAc (150 mL) and washed 3× with saturated NaHCO₃. Theorganic phase was evaporated and the residue purified by silica gelcolumn chromatography using EtOAc-MeOH (95:5) as the eluent, yielding3.85 g (78.3%) of product.

vi.N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoro-methylsulfonyl-1′-D-arabinofuranosyl)guanine

N²-Isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-arabinofuranosyl)guanine(3.84 g) was dissolved in anhydrous CH₂Cl₂ (79 mL), anhydrous pyridine(5 mL) and DMAP (2.93 g) at room temperature under argon. The solutionwas cooled to 0° C. and trifluoromethanesulfonic anhydride (1.99 mL) wasslowly added with stirring. The reaction mixture was stirred at roomtemperature for 1 hour then poured into 100 mL of saturated NaHCO₃. Theaqueous phase was extracted three times with cold CH₂Cl₂. The organicphase was dried over MgSO₄, evaporated and coevaporated with anhydrousMeCN to yield a crude product.

vii.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethylsulfonyl-β-D-ribofuranosyl)guanine.

CrudeN²-isobutyryl-9-(3′,5′-di-O-[tetrahydropyran-2-yl]-2′-O-trifluoromethyl-sulfonyl-β-D-arabinofuranosyl)guaninewas dissolved in anhydrous THF (113 mL) under argon at 0° C. 1 M(nBu)₄N⁺F⁻ (dried by coevaporation with pyridine) in THF (36.95 mL) wasadded with stirring. After 1 hour, a further aliquot of (nBu)₄N⁺F⁻ inTHF (36.95 mL) was added. The reaction mixture was stirred at 0° C. for5 hours and stored overnight at −30° C. The reaction mixture wasevaporated under reduced pressure and the residue dissolved in CH₂Cl₂(160 mL) and extracted five times with deionized water. The organicphase was dried over MgSO₄ and evaporated. The residue was purified bysilica gel column chromatography using EtOAc-MeOH (95:5) to yield 5.25 gof product.

viii. N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)guanine

N²-isobutyryl-9-(2′-deoxy-2′-fluoro-3′,5′-di-O-[tetrahydropyran-2-yl]-β-D-ribofuranosyl)guanine (3.85 g) was dissolved in MeOH (80 mL) at room temperature.Pre-washed Dowex 50W resin (12.32 cm³) was added and the reactionmixture stirred at room temperature for 1 hour. The resin was filteredand the filtrate evaporated to dryness. The resin was washed withpyridine-triethylamine-H₂O (1:3:3) until filtrate was clear. Thisfiltrate was evaporated to obtain an oil. The residues from bothfiltrates were combined in H₂O (200 mL) and washed with CH₂Cl₂ (3×100mL). The aqueous phase was evaporated to dryness and the residuerecrystallized from hot MeOH to yield 0.299 g of product as a whitepowder. The remaining MeOH solution was purified by silica gel columnchromatography to further yield 0.783 g of product by elution withEtOH-MeOH (4:1).

ix.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4-dimethoxytrityl]-β-D-ribofuranosyl)guanine

N²-isobutyryl-9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)guanine (1.09 g)was dissolved in pyridine (20 mL) and triethylamine (0.56 mL) at roomtemperature under argon. 4,4′-Dimethoxytrityl chloride (1.20 g, 1.15molar eq.) was added and the reaction mixture stirred at roomtemperature for 5 hours. The mixture was transferred to a separatoryfunnel and extracted with diethyl ether (100 mL). The organic phase waswashed with saturated NaHCO₃ (3×70 mL), and the aqueous phaseback-extracted three times with diethyl ether. The combined organicphases were dried over MgSO₄ and triethylamine (4 mL) was added tomaintain the solution at basic pH. The solvent was evaporated and theresidue purified by silica gel column chromatography usingEtOAc-triethylamine (100:1) and then EtOAc-MeOH-triethylamine (95:5:1)as eluents yielding 1.03 g of product.

x.N²-Isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4-dimethoxy-trityl]-guanosine-3′-O—N,N-diisopropyl-β-D-cyanoethylphosphoramidite

N²-isobutyryl-9-(2′-deoxy-2′-fluoro-5′-O-[4,4′-dimethoxytrityl])-β-D-ribofuranosyl)guanine(0.587 g) was dissolved in anhydrous CH₂Cl₂ (31 mL) anddiisopropylethylamine (0.4 mL) at room temperature under argon. Thesolution was cooled to 0° C. andchloro(diisopropylamino)-β-cyanoethoxyphosphine (0.42 mL) was slowlyadded. The reaction mixture was allowed to warm to room temperature andstirred for 3.5 hours. CH₂Cl₂-triethylamine (100:1, 35 mL) was added andthe mixture washed with saturated NaHCO₃ (6 mL). The organic phase wasdried over MgSO₄ and evaporated under reduced pressure. The residue waspurified by silica gel column chromatography usinghexane-EtOAc-triethylamine (75:25:1) for 2 column volumes, thenhexane-EtOAc-triethylamine (25:75:1), and finally EtOAc-triethylamine.The product-containing fractions were pooled and the solvent evaporatedunder reduced pressure. The resulting oil was coevaporated twice withMeCN and dried under reduced pressure. The resulting white solid wasdissolved in CH₂Cl₂ (3 mL) and dripped into stirring hexane (300 mL).The resulting precipitate was filtered and dried under reduced pressureto yield 0.673 g (88%) of product.

Example 5

Nucleoside amidites having substitution on their sugar and their basefragments are shown in Examples 5-a through 5-k.

Example 5-a Other Nucleoside Amidites i.1-(2-Fluoro-β-D-erythro-pentofuranosyl)-5-methyluridine

2,2′-Anhydro-[1-β-D-arabinofuranosyl)-5-methyluridine] (71 g, 0.32 mmol)(from Example 2-a) and dioxane (700 mL) are placed in a 2 literstainless steel bomb and HF/pyridine (100 g, 70%) was added. The mixturewas heated for 16 hours at 120-[25° C. and then cooled in an ice bath.The bomb was opened and the mixture was poured onto 3 liters of ice. Tothis mixture was added cautiously sodium hydrogen carbonate (300 g) andsaturated sodium bicarbonate solution (400 mL). The mixture was filteredand the filter cake was washed with water (2×100 mL) and methanol (2×500mL). The water and methanol washes were concentrated to dryness invacuo. Methanol (200 mL) and coarse silica gel (80 g) were added to theresidue and the mixture was concentrated to dryness in vacuo. Theresulting material was concentrated onto the silica gel and purified bysilica gel column chromatography using a gradient of ethyl acetate andmethanol (100:0 to 85:15). Pooling and concentration of the productfractions gave 36.9 g (51%, 2 step yield) of the title compound.

Also isolated from this reaction was1-(2-phenyl-β-D-erythro-pentofuranosyl)-5-methyluridine (10.3 g). Thismaterial is formed from the phenol and its sodium salt from the anhydroreaction above when the bomb reaction is carried out on impure material.When The anhydro material is purified this product is not formed. Theformed 1-(2-phenyl-β-D-erythro-pentofuranosyl)-5-methyluridine wasconverted into its DMT/phosphoramidite using the same reactionconditions as for the 2′-Fluoro material.

ii.1-(5-O-Dimethoxytrityl-2-fluoro-β-D-erythro-pentofuranosyl)-5-methyluridine

1-(2-fluoro-β-D-erythro-pentofuranosyl)-5-methyluridine (31.15 g, 0.12mol) was suspended in pyridine (150 mL) and dimethoxytrityl chloride(44.62 g, 0.12 mol) was added. The mixture was stirred in a closed flaskfor 2 hours and then methanol (30 mL) was added. The mixture wasconcentrated in vacuo and the resulting residue was partitioned betweensaturated bicarbonate solution (500 mL) and ethyl acetate (3×500 ml).The ethyl acetate fractions were pooled and dried over magnesiumsulfate, filtered and concentrated in vacuo to a thick oil. The oil wasdissolved in dichloromethane (100 mL), applied to a silica gel columnand eluted with ethyl acetate:hexane:triethylamine, 60/39/1 increasingto 75/24/1. The product fractions were pooled and concentrated in vacuoto give 59.9 g (89%) of the title compound as a foam.

iii.1-(5-O-Dimethoxytrityl-2-fluoro-3-O—N,N-diisopropylamino-2-cyanoethylphosphite-β-D-erythro-pentofuranosyl)-5-methyluridine

1-(5-O-Dimethoxytrityl-2-fluoro-β-D-erythro-pentofuranosyl)-5-methyluridine(59.8 g, 0.106 mol) was dissolved in dichloromethane and 2-cyanoethylN,N,N′,N′-tetraisopropylphosphorodiamidite (46.9 mL, 0.148 mol) anddiisopropylamine tetrazolide (5.46 g, 0.3 eq.) was added. The mixturewas stirred for 16 hours. The mixture was washed with saturated sodiumbicarbonate (1 L) and the bicarbonate solution was back extracted withdichloromethane (500 mL). The combined organic layers were washed withbrine (1 L) and the brine was back extracted with dichloromethane (100mL). The combined organic layers were dried over sodium sulfate,filtered, and concentrated to a vol of about 200 mL. The resultingmaterial was purified by silica gel column chromatography usinghexane/ethyl acetate/triethyl amine 60/40/1. The product fractions wereconcentrated in vacuo, dissolved in acetonitrile (500 ml), filtered,concentrated in vacuo, and dried to a foam. The foam was chopped anddried for 24 hour to a constant weight to give 68.2 g (84%) of the titlecompound. 1H NMR: (CDCl₃) δ 0.9-1.4 (m, 14H, 4×CH₃, 2×CH), 2.3-2.4 (t,1H, CH₂CN), 2.6-2.7 (t, 1H, CH₂CN), 3.3-3.8 (m, 13H, 2×CH₃OAr, 5′ CH₂,CH₂OP, C-5 CH₃), 4.2-4.3 (m, 1H, 4′), 4.35-5.0 (m, 1H, 3′), 4.9-5.2 (m,1H, 2′), 6.0-6.1 (dd, 1H, 1′), 6.8-7.4 (m, 13H, DMT), 7.5-7.6 (d, 1H,C-6), 8.8 (bs, 1H, NH). ³¹P NMR (CDCl₃); 151.468, 151.609, 151.790,151.904.

iv.1-(3′,5′-di-O-acetyl-2-fluoro-β-D-erythro-pentofuranosyl)-5-methyluridine

1-(2-fluoro-β-D-erythro-pentofuranosyl)-5-methyluridine (22.4 g, 92mmol, 85% purity), prepared as per the procedure of Example 5-a-i., wasazeotroped with pyridine (2×150 mL) and dissolved in pyridine (250 mL).Acetic anhydride (55 mL, 0.58 mol) was added and the mixture was stirredfor 16 hours. Methanol (50 mL) was added and stirring was continued for30 minutes. The mixture was evaporated to a syrup. The syrup wasdissolved in a minimum amount of methanol and loaded onto a silica gelcolumn. Hexane/ethyl acetate, 1:1, was used to elute the productfractions. Purification gave 19.0 g (74%) of the title compound.

Example 5-b i.4-Triazine-1-(3′,5′-di-O-acetyl-2-fluoro-β-D-erythro-pentofuranosyl)-5-methyluridine

1,2,4-Triazole (106 g, 1.53 mol) was dissolved in acetonitrile (150 mL)followed by triethylamine (257 mL, 1.84 mol). The mixture was cooled tobetween 0 and 10° C. using an ice bath. POCl₃ (34.5 mL, 0.375 mol) wasadded slowly via addition funnel and the mixture was stirred for anadditional 45 minutes. In a separate flask,1-(3′,5′-Di-O-acetyl-2-fluoro-β-D-erythro-1-pentofuranosyl)-5-methyluridine(56.9 g, 0.144 mol) was dissolved in acetonitrile (150 mL). The solutioncontaining the1-(3′,5′-Di-O-acetyl-2-fluoro-β-D-erythro-pentofuranosyl)-5-methyluridinewas added via cannula to the triazole solution slowly. The ice bath wasremoved and the reaction mixture was allowed to warm to room temperaturefor 1 hour. The acetonitrile was removed in vacuo and the residue waspartitioned between saturated sodium bicarbonate solution (400 mL) anddichloromethane (4×400 mL). The organic layers were combined andconcentrated in vacuo. The resulting residue was dissolved in ethylacetate (200 mL) and started to precipitate a solid. Hexanes (300 mL)was added and additional solid precipitated. The solid was collected byfiltration and washed with hexanes (2×200 mL) and dried in vacuo to give63.5 g which was used as is without further purification.

ii. 5-methyl-1-(2-fluoro-β-D-erythro-pentofuranosyl)-cytosine

4-Triazine-1-(3′,5′-di-O-acetyl-2-fluoro-β-D-erythro-pentofuranosyl)-thymine(75.5 g, 0.198 mol) was dissolved in ammonia (400 mL) in a stainlesssteel bomb and sealed overnight. The bomb was cooled and opened and theammonia was evaporated. Methanol was added to transfer the material to aflask and about 10 volumes of ethyl ether was added. The mixture wasstirred for 10 minutes and then filtered. The solid was washed withethyl ether and dried to give 51.7 g (86%) of the title compound.

iii.4-N-Benzoyl-5-methyl-1-(2-fluoro-β-D-erythro-pentofuranosyl)cytosine

5-Methyl-1-(2-fluoro-β-D-erythro-pentofuranosyl)-cytosine (54.6 g, 0.21mol) was suspended in pyridine (700 mL) and benzoic anhydride (70 g,0.309 mol) was added. The mixture was stirred for 48 hours at roomtemperature. The pyridine was removed by evaporation and methanol (800mL) was added and the mixture was stirred. A precipitate formed whichwas filtered, washed with methanol (4×50 mL), washed with ether (3×100mL), and dried in a vacuum oven at 45° C. to give 40.5 g of the titlecompound. The filtrate was concentrated in vacuo and treated withsaturated methanolic ammonia in a bomb overnight at room temperature.The mixture was concentrated in vacuo and the resulting oil was purifiedby silica gel column chromatography. The recycled starting material wasagain treated as above to give an additional 4.9 g of the title compoundto give a combined 45.4 g (61%) of the title compound.

iv.4-N-Benzoyl-5-methyl-1-(2-fluoro-5-O-dimethoxytrityl-βD-erythro-pentofuranosyl)cytosine

4-N-Benzoyl-5-methyl-1-(2-fluoro-β-D-erythro-pentofuranosyl)-cytosine(45.3 g, 0.124 mol) was dissolved in 250 ml dry pyridine anddimethoxytrityl chloride (46.4 g, 0.137 mol) was added. The reactionmixture was stirred at room temperature for 90 minutes and methanol (20mL) was added. The mixture was concentrated in vacuo and partitionedbetween ethyl acetate (2×1 L) and saturated sodium bicarbonate (1 L).The ethyl acetate layers were combined, dried over magnesium sulfate andevaporated in vacuo. The resulting oil was dissolved in dichloromethane(200 mL) and purified by silica gel column chromatography using ethylacetate/hexane/triethyl amine 50:50:1. The product fractions were pooledconcentrated in vacuo dried to give 63.6 g (76.6%) of the titlecompound.

v.4-N-Benzoyl-5-methyl-1-(2-fluoro-3-O—N,N-diisopropylamino-2-cyanoethylphosphite-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)cytosine

4-N-Benzoyl-5-methyl-1-(2-fluoro-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-cytosine(61.8 g, 92.8 mmol) was stirred with dichloromethane (300 mL),2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (40.9 mL, 0.130mol) and diisopropylamine tetrazolide (4.76 g, 0.3 eq.) at roomtemperature for 17 hours. The mixture was washed with saturated sodiumbicarbonate (1 L) and the bicarbonate solution was back extracted withdichloromethane (500 mL). The combined organic layers were washed withbrine (1 L) and the brine was back extracted with dichloromethane (100mL). The combined organic layers were dried over sodium sulfate,filtered, and concentrated to a vol of about 200 mL. The resultingmaterial was purified by silica gel column chromatography usinghexane/ethyl acetate/triethyl amine 60/40/1. The product fractions wereconcentrated in vacuo, dissolved in acetonitrile (500 ml), filtered,concentrated in vacuo, and dried to a foam. The foam was chopped anddried for 24 hours to a constant weight to give 72.4 g (90%) of thetitle compound. 1H NMR: (CDCl₃) δ 1.17-1.3 (m, 12H, 4×CH₃), 1.5-1.6 (m,2H, 2×CH), 2.3-2.4 (t, 1H, CH₂CN), 2.6-2.7 (t, 1H, CH₂CN), 3.3-3.9 (m,13H, 2×CH₃OAr, 5′ CH₂, CH₂OP, C-5 CH₃), 4.2-4.3 (m, 1H, 4′), 4.3-4.7 (m,1H, 3′), 5.0-5.2 (m, 1H, 2′), 6.0-6.2 (dd, 1H, 1′), 6.8-6.9 (m, 4H,DMT), 7.2-7.6 (m, 13H, DMT, Bz), 7.82-7.86 (d, 1H, C-6), 8.2-8.3 (d, 2H,Bz). ³¹P NMR (CDCl₃); bs, 151.706; bs, 151.941.

Example 5-c i.1-(2,3-di-O-Butyltin-β-D-erythro-pentofuranosyl)-5-methyluridine

5-Methyluridine (7.8 g, 30.2 mmol) and dibutyltin oxide (7.7 g, 30.9mmol) were suspended in methanol (150 mL) and heated to reflux for 16hours. The reaction mixture was cooled to room temperature, filtered,and the solid washed with methanol (2×150 mL). The resulting solid wasdried to give 12.2 g (80.3%) of the title compound. This material wasused without further purification in subsequent reactions. NMR wasconsistent with structure.

ii. 1-(2-O-Propyl-β-D-erythro-pentofuranosyl)-5-methyluridine

1-(2,3-di-O-butyltin-β-D-erythro-pentofuranosyl)-5-methyluridine (5.0 g,10.2 mmol) and iodopropane (14.7 g, 72.3 mmol) were stirred in DMF at100° C. for 2 days. The reaction mixture was cooled to room temperatureand filtered and concentrated. The residual DMF was coevaporated withacetonitrile. After drying the residue there was obtained 2.40 g (78%)of the title compound and the 3′-O-propyl isomer as a crude mixture.This material was used without further purification in subsequentreactions.

iii.1-(2-O-Propyl-5-O-Dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methyluridine

1-(2-O-Propyl-β-D-erythro-pentofuranosyl)-5-methyluridine and the3′-O-propyl isomer as a crude mixture (2.4 g, 8.4 mmol) was coevaporatedwith pyridine (2×40 mL) and dissolved in pyridine (60 mL). The solutionwas stirred at room temperature under argon for 15 minutes anddimethoxytrityl chloride (4.27 g, 12.6 mmol) was added. The mixture waschecked periodically by tlc and at 3 hours was completed. Methanol (10mL) was added and the mixture was stirred for 10 minutes. The reactionmixture was concentrated in vacuo and the resulting residue purified bysilica gel column chromatography using 60:40 hexane/ethyl acetate with1% triethylamine used throughout. The pooling and concentration ofappropriate fractions gave 1.32 g (26%) of the title compound.

iv.1-(2-O-Propyl-3-O—N,N-Diisopropylamino-2-cyanoethylphosphite-5-O-Dimethoxytrityl-β-D-erythro-Pentofuranosyl)-5-methyluridine

1-(2-O-Propyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methyluridine(50.0 g, 86 mmol),2-cyanoethyl-N,N,N′,N′-tetra-isopropylphosphorodiamidite (38 mL, 120mmol), and diisopropylamine tetrazolide (4.45 g, 25.8 mmol) weredissolved in dichloromethane (500 mL) and stirred at room temperaturefor 40 hours. The reaction mixture was washed with saturated sodiumbicarbonate solution (2×400 mL) and brine (1×400 mL). The aqueous layerswere back extracted with dichloromethane. The dichloromethane layerswere combined, dried over sodium sulfate, filtered, and concentrated invacuo. The resultant residue was purified by silica gel columnchromatography using ethyl acetate/hexane 40:60 and 1% triethylamine.The appropriate fractions were pooled, concentrated, and dried underhigh vacuum to give 43 g (67%).

v.1-(2-O-Propyl-3-O-acetyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methyluridine

1-(2-O-Propyl-5-dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methyluridine(10.0 g, 16.6 mmol) was dissolved in pyridine (50 mL) and aceticanhydride (4.7 ml, 52.7 mmol) was added. The reaction mixture wasstirred for 18 hours and excess acetic anhydride was neutralized withmethanol (10 mL). The mixture was concentrated in vacuo and theresulting residue dissolved in ethyl acetate (150 mL). The ethyl acetatewas washed with saturated NaHCO₃ (150 mL) and the saturated NaHCO₃ washwas back extracted with ethyl acetate (50 mL). The ethyl acetate layerswere combined and concentrated in vacuo to yield a white foam 11.3 g.The crude yield was greater than 100% and the NMR was consistent withthe expected structure of the title compound. This material was usedwithout further purification in subsequent reactions.

Example 5-d i.1-(2-O-Propyl-3-O-acetyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-4-triazolo-5-methylpyrimidine

Triazole (10.5 g, 152 mmol) was dissolved in acetonitrile (120 ml) andtriethylamine (23 mL) with stirring under anhydrous conditions. Theresulting solution was cooled in a dry ice acetone bath and phosphorousoxychloride (3.9 mL, 41 mmol) was added slowly over a period of 5minutes. The mixture was stirred for an additional 10 minutes becoming athin slurry indicative of product formation.1-(2-O-Propyl-3-O-acetyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methyluridine(11.2 g, 165 mmol) was dissolved in acetonitrile (150 mL) and added tothe slurry above, maintaining dry ice acetone bath temperatures. Thereaction mixture was stirred for 30 minutes and then allowed to warm toroom temperature and stirred for an additional 2 hours. The mixture wasplaced in a freezer at 0° C. for 18 hours and then removed and allowedto warm to room temperature. Tlc in ethyl acetate/hexane 1:1 of themixture showed complete conversion of the starting material. Thereaction mixture was concentrated in vacuo and redissolved in ethylacetate (300 mL) and extracted with saturated sodium bicarbonatesolution (2×400 mL) and brine (400 mL). The aqueous layers were backextracted with ethyl acetate (200 mL). The ethyl acetate layers werecombined, dried over sodium sulfate, and concentrated in vacuo. Thecrude yield was 11.3 g (95%). The NMR was consistent with the expectedstructure of the title compound. This material was used without furtherpurification in subsequent reactions.

ii.1-(2-O-Propyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methylcytidine

1-(2-O-Propyl-3-O-acetyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-4-triazolo-5-methylpyrimidine(11.2 g, 16.1 mmol) was dissolved in liquid ammonia (50 mL) in a 100 mLbomb at dry ice acetone temperatures. The bomb was allowed to warm toroom temperature for 18 hours and then recooled to dry ice acetonetemperatures. The bomb contents were transferred to a beaker andmethanol (50 mL) was added. The mixture was allowed to evaporate to neardryness. Ethyl acetate (300 mL) was added and some solid was filteredoff prior to washing with saturated sodium bicarbonate solution (2×250mL). The ethyl acetate layers were dried over sodium sulfate, filtered,combined with the solid previously filtered off, and concentrated invacuo to give 10.1 g of material. The crude yield was greater than 100%and the NMR was consistent with the expected structure of the titlecompound. This material was used without further purification insubsequent reactions.

iii.1-(2-O-Propyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-4-N-benzoyl-5-methylcytidine

1-(2-O-Propyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methylcytidine(7.28 g, 10.1 mmol) and benzoic anhydride (4.5 g, 20 mmol) weredissolved in DMF (60 mL) and stirred at room temperature for 18 hours.The reaction mixture was concentrated in vacuo and redissolved in ethylacetate (300 mL). The ethyl acetate solution was washed with saturatedsodium bicarbonate solution (2×400 mL), dried over sodium sulfate,filtered, and concentrated in vacuo. The residue was purified by silicagel column chromatography using ethyl acetate/hexane 1:2 and 1%triethylamine. The appropriate fractions were pooled, concentrated, anddried under high vacuum to give 5.1 g (59% for 4 steps starting with the1-(2-O-propyl-dimethoxytrityl-β-D-erythro-pentofuranosyl)-5-methyluridine).

iv.1-(2-O-Propyl-3-O—N,N-diisopropylamino-2-cyanoethyl-phosphite-5-O-dimethoxytrityl-β-D-ethythro-pentofuranosyl)-4-N-benzoyl-5-methylcytidine

1-(2-O-Propyl-5-O-dimethoxytrityl-β-D-erythro-pentofuranosyl)-4-N-benzoyl-5-methylcytidine(5.0 g, 7 mmol),2-cyanoethyl-N,N,N′,N′-tetra-isopropylphosphorodiamidite (3.6 mL, 11.3mmol), and diisopropylaminotetrazolide (0.42 g, 2.4 mmol) were dissolvedin dichloromethane (80 mL) and stirred at room temperature for 40 hours.The reaction mixture was washed with saturated sodium bicarbonatesolution (2×40 mL) and brine (1×40 mL). The aqueous layers were backextracted with dichloromethane. The dichloromethane layers werecombined, dried over sodium sulfate, filtered, and concentrated invacuo. The resultant residue was purified by silica gel columnchromatography using ethyl acetate/hexane 40:60 and 1% triethylamine.The appropriate fractions were pooled, concentrated, and dried underhigh vacuum to give 7.3 g (98%).

Example 5-e i. 2′-O-Methyl-5-methyluridine Procedure 1:

Crude 2,2′-anhydro-5-methyluridine (10.0 g, 0.0416 mol) (Example 2-a)was dissolved in methanol (80 mL) in a stainless steel bomb (100 mLcapacity). Trimethyl borate (5.6 mL, 0.049 mol) was added (Note 1). Thebomb was sealed and placed in an oil bath at 150° C. which generated apressure of about 5 atm. After 40 h, the bomb was cooled in ice, openedand the contents concentrated under reduced pressure to a tan foam, 12g. NMR of the crude was consistent with the product contaminated withimpurities in the starting material and a trace of thymine and startingmaterial (Note 2). The crude product was used as is for the next step.

The trialkyl borates can be conveniently generated by adding solutions(eg 1 M in THF) of borane to the desired alcohol and allowing theresulting hydrogen gas to evolve.) The nucleoside can be purified atthis point by column chromatography using a gradient of methanol inethyl acetate (0-10%) and crystallizing the product from absoluteethanol to give white needles, mp 192-193° (mp) 197-198°. Literaturereference for the melting point of this compound is contained in E.Ootsuka, H. Inoue, Japanese Patent 89-85456, 4 Apr. 1989.

Procedure 2:

Pure 2,2′-anhydro-5-methyluridine (1.0 g, 4.16 mmol) and trimethylborate(0.56 mL, 4.9 mmol) was dissolved in methanol (20 mL) in a stainlesssteel bomb (100 mL). The bomb was placed in an oil bath at 150° C. After80 h, TLC indicating the reaction to be mostly complete. The solvent wasremoved yielding a white foam. NMR indicated product to startingmaterial ratio of 93:7 with no other impurities noted. The residue waspurified by silica gel column chromatography using a methanol gradientin ethyl acetate (0-10%) yielding 850 mg (75%) of pure product and 250mg of still contaminated product. An analytically pure sample wasprepared for NMR. ¹H NMR (DMSO-d₆): δ 1.79 (s, 3H, 5-CH₃), 3.35 (s, 3H,OCH₃), 3.5-3.7 (m, 2H, H-5′), 3.7-3.9 (m, 2H, H-3′,4′), 4.15 (m, 1H,H-2′), 5.17 (m, 2H, 3′,5′-OH), 5.87 (d, J=5 Hz, 1H, H-1′), 7.80 (s, 1H,H-6), 11.37 (br s, 1H, N—H). Anal. Calcd for C₁₁H₁₆N₂O₆ (272.26): C,48.52; H, 5.92; N, 10.29. Found: C, 48.56; H, 5.88; N, 10.22.

Procedure 3:

The same as described for procedure 2 except 30 mg of sodium bicarbonatewas added to the reaction (to match the sodium content of the crudeanhydro) which allowed the reaction to be complete in 24 h. Ammoniumchloride (50 mg) was added to neutralize the base and the solution wasstripped to dryness. NMR of the crude indicated three minor nucleosideimpurities (total about 6%). After a similar column and thencrystallizing the residue from methanol/ethyl acetate, there remained850 mg of first crop material and 120 mg of second crop material bothwith 2-3% of unknown nucleoside impurities for a still contaminatedyield of 85%.

ii. 5′-O-Dimethoxytriphenylmethyl-2′-O-methyl-5-methyluridine

Crude 2′-O-methyl-5-methyl uridine (12 g) was coevaporated in pyridine(2×50 mL) and dissolved in dry pyridine (50 mL).Dimethoxytriphenylmethyl chloride (18.1 g, 0.054 mol) was added. Theflask was stoppered and allowed to stand for 45 min at room temperature.Methanol (10 mL) was added to quench the reaction and the solution wasconcentrated under reduced pressure to an oil. The residue waspartitioned between ethyl acetate (2×400 mL) and saturated sodiumbicarbonate solution (500 mL). The organic layers were combined, dried(sodium sulfate), filtered and concentrated to a yellow foam. The foamwas dissolved in methylene chloride (60 mL) and put onto a silica gelcolumn (300 g) and eluted with ethyl acetate-hexanes-triethylamine,60:40:1. The product containing fractions were combined, concentratedand coevaporated with dry acetonitrile (2×50 mL). The resulting residuewas dried at 1 mm Hg for 24 h to a crisp white foam, 17.0 g (60.4% inthree steps from 5-methyluridine).

Example 5-f i. 2,3,5-Tri-O-benzoyl-2-thio-5-methyluridine

In a 250 ml 3 neck round bottomed flask 1-O-acetyl-2,3,5-tri-O-benzoylribose (0.500 g, 1 mmol) and 5-methyl-2-thiouracil (0.156 g, 1.1 mmol)was dried under vacuum overnight. These components were dissolved in 10mL of dry acetonitrile and heated to 80° C. To this warm solution wasadded N—O-bis(trimethylsilyl)acetamide (0.509 g, 2.5 mmol) and thereaction stirred for 1 hr at 80° C. The reaction mixture was removedfrom the heat and allowed to cool to room temperature, and trimethylsilyl triflate (0.334 g, 1.5 mmol) was added dropwise. The reactionmixture was then heated to 50° C. and stirred for 4 hours. The reactionmixture was checked by TLC using ethyl acetate/hexane 1:1, which showedthe reaction had gone to completion. The solution was cooled to roomtemperature and partitioned between 50 mL of dichloromethane and 50 mLof saturated sodium bicarbonate solution. The aqueous phase wasextracted two more times with dichloromethane and the organic layerscombined, dried with magnesium sulfate and concentrated to a pale yellowfoam. This foam was used without further purification.

ii. 2-Thio-5-methyluridine

The crude 2,3,5-tri-O-benzoyl-2-thio-5-methyl uridine (20 g, 37 mmoles)was dissolved in 500 mL of methanol. To this solution was added sodiummethoxide (2.0 g, 37 mmoles) and the reaction stirred for 2 hours. Thereaction was checked by TLC using ethyl acetate/hexane 1:1 and ethylacetate/methanol 9:1, which showed the reaction had gone to completion.Dowex 50H⁺ resin was added until the solution was neutral by pH paperand the resin filtered. The resin was then washed with 100 ml ofadditional methanol and the combined filtrates were concentrated to givethe title compound 8.5 g, (84%) as a pale yellow foam.

Example 5-g 2′-O-Methyl-5-methyl-2-thiouridine

To a stirred solution of 5-methyl-2-thiouridine (0.500 g, 1.8 mmol) inDMF (10 ml) is added dibutyltin oxide (0.500 g, 2.0 mmol), tetrabutylammonium iodide (0.738 g, 2 mmol), and methyl iodide (1.022 g, 7.2mmol). The reaction flask is sealed and heated at 50° C. for 16 hours.The mixture is cooled and another portion of methyl iodide is added(1.022 g, 7.2 mmol) and the reaction heated for an additional 16 hours.At the end of this time, the reaction mixture is cooled to roomtemperature and diluted with methylene chloride and chromatographedusing a methylene chloride/methanol gradient. The appropriate fractionsare collected and concentrated to give2′-O-methyl-5-methyl-2-thiouridine.

Example 5-h 2′-O-Propyl-5-methyl-2-thiouridine

The title compound is prepared as per the procedures of Example 5-g bysubstituting propyl iodide (1.22 g, 7.2 mmoles) in place of methyliodide.

Example 5-i i. 2′-O-phthalimidopropyl-5-methyl-2-thiouridine

The title compound was prepared as per the procedures of Example 5-g bysubstituting bromo-propyl phthalimide (0.67 g, 2.5 mmoles) in place ofmethyl iodide, with an additional (0.300 g) added on the second day.

ii. 5′-O-Dimethoxytrityl-2′-O-propylamine-5-methyl-2-thiouridine

2′-O-Phthalimidopropyl-5-methyl-2-thiouridine (2.6 g, 3.6 mmol) wasdissolved in dry pyridine and co-evaporated twice. The resulting foamwas dissolved in 25 mL of dry pyridine and dimethoxy-trityl chloride(1.8 g, 5.5 mmol) was added followed by 4,4-dimethylaminopyridine (0.050g, 0.4 mmol). The reaction was allowed to stir overnight at roomtemperature. To the reaction mixture was added 1 mL of methanol. Thesolution was partitioned between 75 mL of saturated sodium bicarbonateand 50 mL of chloroform. The aqueous layer was extracted with twoadditional portions of chloroform and the organic layers combined anddried with magnesium sulfate. After removal of the drying agent viafiltration the filtrate was concentrated to an orange oil and purifiedby silica gel column chromatography using methanol/chloroform gradientwith 0.5% pyridine added to neutralize the silica gel.

iii.5′-O-Dimethoxytrityl-2′-O-propylamine-5-methyl-2S-toluoyl-2-thiouridine

5′-O-Dimethoxytrityl-2′-O-propylamine-5-methyl-2-thiouridine (1 g, 1.6mmol) was dissolved in DMF and cooled to 0° C. To this solution wasadded triethyl amine (0.300 g, 3 m.mol) followed by toluoyl chloride(0.300 g, 1.92 mmol) dropwise over 5 minutes. The reaction was thenallowed to warm to room temperature and stirred overnight, when completethe reaction was quenched with methanol and concentrated to an oil. Theoil was then partitioned between 250 mL of a solution of saturatedsodium bicarbonate/chloroform 1:1. The aqueous layer was extracted withtwo additional, 75 mL portions of chloroform, and the organic layerswere dried and concentrated to an oil. The protected nucleoside waspurified by silica gel column chromatography using a hexane/ethylacetate gradient. The desired product was collected as a mixture of N-3toluoyl and S-2 Toluoyl compounds. This mixture was used as is for thephosphyt-ilation procedure.

iv.5′-O-Dimethoxytrityl-T-O-propylamine-3′-O—[(N,N-diisopropylamino)-2-cyanoethoxyphosphite]-5-methyl-2-S-toluoyl-2-thiouridine

To a solution of5′-O-dimethoxytrityl-2′-O-propylamine-5-methyl-2-S-toluoyl-2-thiouridine(16.01 g, 22 mmol) and diisopropylethylamine (10 ml) in THF (200 ml), at0° C., is added chloro-β-cyanoethoxy-N,N-diisopropylaminophosphine (5.6ml, 25 mmol). The reaction mixture was stirred at room temperature for20 hours. The reaction was concentrated and the residue purified bysilica gel column chromatography. Elution with an ethyl acetate/hexanegradient while maintaining 1% triethylamine, pooling of appropriatefractions and evaporation will give the title compound.

Example 5-j i. 2′-O-Aminopropyl-5-methyl-2-thiouridine

2′-O-Phthalimidopropyl-5-methyl-2-thiouridine (5.0 g, 15.8 mmol) isdissolved in 100 ml methanol in a 500 ml flask. Hydrazine (2.02 g, 63.2mmol) is added and the mixture is heated to reflux (60-65° C.) withstirring for 14 hours. The solvent is evaporated in vacuo and theresidue is dissolved in dichloromethane (150 ml) and extracted twicewith an equal volume NH₄OH. The organic layer is evaporated to yield thecrude product. NMR is used to assay product purity. The product is usedin subsequent reactions without further purification.

ii. 2′-O-Trifluoroacetylaminopropyl-5-methyl-2-thiouridine

2′-O-Aminopropyl-5-methyl-2-thiouridine is dissolved in MeOH and 5equivalents of triethylamine are added followed by 10 equivalents ofethyl trifluoroacetate. The title compound is isolated afterpurification.

iii.2′-O-Trifluoroacetylaminopropyl-5′-O-dimethoxytrityl-5-methyl-2-thiouridine

2′-O-Trifluoroacetylaminopropyl-5-methyl-2-thiouridine (2.5 g, 3.6 mmol)is dissolved in dry pyridine and co-evaporated twice. The resultingyellow foam is dissolved in 25 mL of dry pyridine and dimethoxytritylchloride (1.8 g, 5.5 mmol) is added followed by4,4-dimethylaminopyridine (0.050 g, 0.4 mmol). The reaction is allowedto stir overnight at room temperature. To the reaction mixture is added1 mL of methanol. The solution is partitioned between 75 mL of saturatedsodium bicarbonate and 50 mL of chloroform. The aqueous layer isextracted with two additional portions of chloroform and the organiclayers combined and dried with magnesium sulfate. After removal of thedrying agent via filtration the filtrate is concentrated to an oil andpurified by silica gel column chromatography using methanol/chloroformgradient with 0.5% pyridine added to neutralize the silica gel to givethe title compound.

iv.2′-O-Trifluoroacetylaminopropyl-3′-O—[(N,N-diisopropylamino)-2-cyanoethoxyphosphite]-5′-O-dimethoxytrityl-5-methyl-2-thiouridine

The title compound is prepared as per the procedure of Example 5-i-iv.using the title compound from Example 5-j-iii.

Example 5-k i. 5′-O-Dimethoxytrityl-2-thio-5-methyluridine

2-Thio-5-methyl uridine (1 g, 3.6 mmol) was dissolved in dry pyridineand co-evaporated twice. The resulting yellow foam was dissolved in 25mL of dry pyridine and dimethoxy-trityl chloride (1.8 g, 5.5 mmol) wasadded followed by 4,4-dimethylaminopyridine (0.050 g, 0.4 mmol). Thereaction was allowed to stir overnight at room temperature. To thereaction mixture was added 1 mL of methanol. The solution waspartitioned between 75 mL of saturated sodium bicarbonate and 50 mL ofchloroform. The aqueous layer was extracted with two additional portionsof chloroform and the organic layers combined and dried with magnesiumsulfate. After removal of the drying agent via filtration the filtratewas concentrated to an orange oil and purified by silica gel columnchromatography using methanol/chloroform gradient with 0.5% pyridineadded to neutralize the silica gel.

ii. 5′-O-Dimethoxytrityl-3′-t-butyldimethylsilyl-5-methyl-2-thiouridine

5′-O-Dimethoxytrityl-2-thio-5-methyl uridine (1 g, 1.73 mmol) wasco-evaporated twice with dry DMF and then dissolved in dry DMF andimidazole (0.141 g, 2.08 mmol) was added followed by (0.313 g, 2.08mmol) of t-butyl-dimethylsilyl chloride. The reaction mixture wasstirred overnight. The reaction was checked by TLC using ethylacetate/hexane 1:1, which showed the reaction had gone to completion.The reaction mixture was then poured into 5% sodium bicarbonate andextracted 3 times with chloroform. The combined organic solution wasdried with magnesium sulfate and concentrated to an oil. The resultingoil was purified by silica gel column chromatography using amethanol/chloroform gradient isolating separately the 2′ and 3′ silylprotected nucleoside.

iii.5′-O-Dimethoxytrityl-3′-t-butyldimethylsilyl-2′-methanesulfonyl-5-methyl-2-thiouridine

5′-O-Dimethoxytrityl-3′-t-butyldimethylsilyl-5-methyl-2-thiouridine (1.0g, 1.45 mmoles) was dissolved in pyridine and cooled to 0° C. To thissolution was added methanesulfonyl chloride (0.183 g, 1.6 mmoles)dropwise. The reaction was then allowed to stir until complete by TLC.The reaction mixture is neutralized with methanol and concentrated to anoil. The title compound is used as is for further reactions.

iv. 5′-Dimethoxytrityl-3′-t-butyldimethylsilyl-2,2-thioanhydro-5-methyl-2-thiouridine

The mesylated nucleoside found in Example 5-k-iii is treated at roomtemperature with 5 equivalents of sodium methoxide and allowed to stiruntil complete formation of the thioanhydro product. The solution isthen neutralized with Dowex 50W (H⁺ form), the resin filtered off andthe resulting solution concentrated to give the title compound.

v.2′-Fluoro-3′-t-butyldimethylsilyl-5′-Dimethoxytrityl-5-methyl-2-thiouridine

The thioanhydronucleoside found in Example 5-k-iv was dissolved inanhydrous dioxane. To this solution was added 6 equivalents ofHF/Pyridine complex and the reaction stirred until complete by TLC. Thereaction mixture is then poured over an equal volume of ice and calciumcarbonate is added until neutral. The solids are filtered off and thefiltrate is concentrated. The residue is purified by silica gel columnchromatography to give the title compound.

vi.2′-Fluoro-3′-O—[(N,N-diisopropylamino)-2-cyanoethoxyphosphite]-5′-dimethoxytrityl-5-methyl-2-thiouridine

2′-Fluoro-3′-t-butyldimethylsilyl-5′-dimethoxytrityl-5-methyl-2-thiouridineis treated as per the procedure of Example 5-i-iv. to give the titlecompound.

Example 6 Oligoribonucleotide Synthesis

Unsubstituted and substituted phosphodiester oligoribonucleotides, alsoidentified herein as PO linked oligoribonucleotides, were synthesized onan automated DNA synthesizer (Applied Biosystems model 380B) usingstandard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioate oligonucleotides, also identified herein as PS linkedoligoribonucleotides, are synthesized as per the phosphodiesteroligoribonucleotides except the standard oxidation bottle was replacedby 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide inacetonitrile for the step wise thiation of the phosphite linkages. Thethiation wait step was increased to 68 sec and was followed by thecapping step. After cleavage from the CPG column and deblocking inconcentrated ammonium hydroxide at 55° C. (18 hr), the oligonucleotideswere purified by precipitating twice with 2.5 volumes of ethanol from a0.5 M NaCl solution. Analytical gel electrophoresis was accomplished in20% acrylamide, 8 M urea, 454 mM Tris-borate buffer, pH=7.0.Oligonucleotides and phosphorothioates were judged, based onpolyacrylamide gel electrophoresis, to be greater than 80% full-lengthmaterial.

Phosphinate oligoribonucleotides, also identified herein as PI linkedoligoribonucleotides, are prepared as is described in U.S. Pat. No.5,508,270, herein incorporated by reference.

Alkyl phosphonate oligoribonucleotides, also identified herein as PMelinked oligoribonucleotides, are prepared as is described in U.S. Pat.No. 4,469,863, herein incorporated by reference.

Phosphoramidite oligoribonucleotides, also identified herein as PNlinked oligoribonucleotides, are prepared as is described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligoribonucleotides, also identified herein asMePS linked oligoribonucleotides, are prepared as is described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporate byreference.

3′-Deoxy-3′-amino phosphoramidate oligoribonucleotide, also identifiedherein as 3′NPN linked oligoribonucleotides, are prepared as isdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligoribonucleotides, also identified herein as POMelinked oligoribonucleotides, are prepared as is described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligoribonucleotide, also identified herein as BPlinked oligoribonucleotides, are prepared as is described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Example 7-a Oligoribonucleoside Synthesis

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages are prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively), herein incorporated byreference.

Formacetal and thioformacetal linked oligoribonucleosides, alsoidentified herein as FA and TFA oligoribonucleosides, respectively, areprepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564,herein incorporated by reference.

Ethylene oxide linked oligoribonucleosides, also herein identified asETO linked oligoribonucleosides, are prepared as is described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 7-b PNA

Peptide Nucleic Acids (PNAs) are known per se and are prepared inaccordance with any of the various procedures referred to in PeptideNucleic Acids (PNA): Synthesis, Properties and Potential Applications,Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also beprepared in accordance with U.S. Pat. No. 5,539,083, corresponding toSer. No. 08/200,742, filed Feb. 23, 1994, and assigned to the sameassignee as this application. These references are herein incorporatedby reference.

Example 8 Chimeric phosphorothioate oligoribonucleotides, e.g.[2′-O-Me]/PS.[2′-OH]/PS.[-2′-O-Me]/PS oligoribonucleotide

Chimeric oligoribonucleotides having 2′-O-alkyl phosphorothioate and2′-OH phosphorothioate oligonucleotides segments were synthesized usingan Applied Biosystems automated DNA synthesizer Model 380B, as above.Oligoribonucleotides were synthesized using the automated synthesizerand 5′-dimethoxytrityl-2′-tert-butyldimethylsilyl 3′-O-phosphoramiditefor the RNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoroamidite for 5′ and 3′wings. The protecting groups on the exocyclic amines were, phenoxyacetylfor rA and rG, benzoyl for rC and 2′-0-methyl A and 2′-O-methyl C, andisobutyryl for 2′-O-methyl G. The standard synthesis cycle was modifiedby increasing the wait step after the delivery of tetrazole and base to600 s repeated four times for RNA and twice for 2′-0-methyl. The fullyprotected oligoribonucleotide was cleaved from the support and thephosphate group was deprotected in 3:1 Ammonia/Ethanol at roomtemperature overnight then lyophilized to dryness. Treatment inmethanolic ammonia for 24 hrs at room temperature was then done todeprotect all bases and sample was again lyophilized to dryness. Thepellet was resuspended in 1M TBAF in THF for 24 hrs at room temperatureto deprotect the 2′ positions. The reaction is then quenched with 1MTEAA and the sample is then reduced to ½ volume by rotovac before beingdesalted on a G25 size exclusion column. The oligo recovered was thenanalyzed spectrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometer.

Example 9 Chimeric “Gapmer” Oligoribonucleotides i. Chimeric MethylPhosphonate Oligoribonucleotide e.g.,[2′-O-Me]/PMe.[2′-OH]/PMe-[-2′-O-Me]/PMe Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a methyl phosphonate backbone isprepared.

ii. Chimeric Phosphoramidate Oligoribonucleotide, e.g.,[2′-O-Me]/PN.[2′-OH]/PN.[-2′-O-Me]/PN Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a phosphoramidate backbone isprepared.

iii. Chimeric Phosphoramidate Oligoribonucleotide, e.g.,[2′-O-Me]/3′NPN.[2′-OH]/3′NPN.[2′-O-Me]/3′NPN Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a 3′-deoxy-3′-amino phosphoramidatebackbone is prepared.

iv. Chimeric Phosphinate Oligoribonucleotide, e.g.,[2′-O-Me]/PI.[2′-OH]/PI.[-2′-O-Me]/PI Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a phosphinate backbone is prepared.

v. Chimeric Alkylphosphonothioate Oligoribonucleotide, e.g.,[2′-O-Me]/MePS.[2′-OH]/MePS.[-2′-O-Me]/MePS Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a phosphonothioate backbone isprepared.

vi. Chimeric Phosphorodithioate Oligoribonucleotide, e.g.,[2′-O-Me]/P2S.[2′-OH]/P2S.[-2′-O-Me]/P2S Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a phosphorodithioate backbone isprepared.

vii. Chimeric Phosphoselenate Oligoribonucleotide, e.g.,[2′-O-Me]/PSe.[2′-OH]/PSe.[-2′-O-Me]/PSe Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a phosphoselenate backbone isprepared.

viii. Chimeric Borano Phosphate Oligoribonucleotide, e.g.,[2′-O-Me]/BP.[2′-OH]/BP.[-2′-O-Me]/BP Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a borano phosphate backbone isprepared.

ix. Chimeric Methyl Phosphotriester Oligoribonucleotide, e.g.,[2′-O-Me]/POME.[2′-OH]/POMe.[-2′-O-Me]/POMe Oligoribonucleotide

In the manner of Example 8, using oligoribonucleotides of Example 6, achimeric oligoribonucleotide having a methyl phosphotriester backbone isprepared.

Example 10 Chimeric Oligoribonucleosides i. ChimericMethylenemethylimino Oligoribonucleoside, e.g.[2′-O-Me]/MMI.[2′-OH]/MMI.[-2′-O-Me]/MMI Oligoribonucleoside

In the manner of Example 8 using the chemistry of Example 7, a chimericoligoribonucleoside having methylenemethylimino linkages throughout theoligoribonucleoside is prepared.

ii. Chimeric Methylenedimethyhydrazo Oligoribonucleoside, e.g.[2′-O-Me]/MDH.[2′-OH]/MDH.[-2′-O-Me]/MDH Oligoribonucleoside

In the manner of Example 8 using the chemistry of Example 7, a chimericoligoribonucleoside having methylenedimethylhydrazo linkages throughoutthe oligoribonucleoside is prepared.

iii. Chimeric Formacetal Oligoribonucleoside, e.g.[2′-O-Me]/FA.[2′-OH]/FA.[-2′-O-Me]/FA Oligoribonucleoside

In the manner of Example 8 using the chemistry of Example 7, a chimericoligoribonucleoside having formacetal linkages throughout theoligoribonucleoside is prepared.

iv. Chimeric Thioformacetal Oligoribonucleoside, e.g.[2-O-Me]/TFA.[2′-OH]/TFA.[-2′-O-Me]/TFA Oligoribonucleoside

In the manner of Example 8 using the chemistry of Example 7, a chimericoligoribonucleoside having thioformacetal linkages throughout theoligoribonucleoside is prepared.

v. Chimeric Ethyleneoxide Oligoribonucleoside, e.g.[2′-O-Me]/ETO.[2′-OH]/ETO.[-2′-O-Me]/ETO Oligoribonucleoside

In the manner of Example 8 using the chemistry of Example 7, a chimericoligoribonucleoside having ethylene oxide linkages throughout theoligoribonucleoside is prepared.

vi. Chimeric methylenecarbonylamino oligoribonucleoside, e.g.[2′-O-Me]/amide-3.[2′-OH]/amide-3.[-2′-O-Me]/amide-3 Oligoribonucleoside

In the manner of Example 8 using the chemistry of Example 7, a chimericoligoribonucleoside having amide-3 linkages throughout theoligoribonucleoside is prepared.

Example 11 Chimeric Oligoribonucleotides/Oligoribonucleosides i.Methylenemethylimino/phosphorothioate chimera, e.g.

[2′-O-Me]/PS.[2′-OH]/PS.[-2′-O-Me]/MMIOligoribonucleotide/Oligoribonucleoside

In the manner of Example 8 using the chemistry of Examples 6 and 7, achimeric compound having both oligoribonucleotide andoligoribonucleoside segments is prepared. The chimeric compounds hasmethylenemethylimino linkages in one “wing” and phosphorothioatelinkages in a central “gap” and in the other “wing.”

ii. Chimeric Methyl Phosphonate/Methylenemethylimino/PhosphorothioateOligoribonucleotide/Oligoribonucleoside, e.g.[2′-O-Me]/PMe.[2′-OH]/PS.[-2′-O-Me]/MMIOligoribonucleotide/Oligoribonucleoside

In the manner of Example 8 using the chemistry of Examples 6 and 7, achimeric compound having both oligoribonucleotide andoligoribonucleoside portions is prepared. The chimeric compound hasmethylenemethylimino linkages in one “wing”, a phosphorothioate linkagesin a central “gap” and methyl phosphonate linkages in the other “wing.”

iii. ChimericMethylenecarbonylamino/Phosphorothioate/MethylenecarbonylaminoOligoribonucleotide/Oligoribonucleoside, e.g.[2′-O-Me]/amide-3.[2′-OH]/PS.[-2′-O-Me]/amide-3Oligoribonucleotide/Oligoribonucleoside

In the manner of Example 8 using the chemistry of Examples 6 and 7, achimeric compound having both oligoribonucleotide andoligoribonucleoside segments is prepared. The chimeric compound hasmethylenecarbonylaimino linkages in both “wings” and phosphorothioatelinkages in a central “gap.”

iv. ChimericMethylenecarbonylamino/Phosphorothioate/MethylenemethyliminoOligoribonucleotide/Oligoribonucleoside, e.g.[2′-O-Me]/amide-3.[2′-OH]/PS.[-2′-O-Me]/MMIOligoribonucleotide/Oligoribonucleoside

In the manner of Example 8 using the chemistry of Examples 6 and 7, achimeric compound having both oligoribonucleotide andoligoribonucleoside segments is prepared. The chimeric compound hasmethylenecarbonylaimino linkages in one “wing” segment, phosphorothioatelinkages in a central “gap” segment and methylenecarbonylamino linkagesin the other “wing” segment.

v. Methylenemethylimino/Phosphodiester/Phosphorothioate Chimera, e.g.[2′-O-Me]/MMI-PO.[2′-OH]/PS.[-2′-O-Me]/MMI-POOligoribonucleotide/Oligoribonucleoside

In the manner of Example 8 using the chemistry of Examples 6 and 7, achimeric compound having both oligoribonucleotide andoligoribonucleoside segments is prepared. The chimeric compounds hasalternating methylenemethylimino and phosphodiester linkages in its“wing” segments and phosphorothioate linkages in its central “gap”segment.

Example 12 Chimeric “end” Gapped Phosphorothioate Oligoribonucleotidesi. “3′-End” Gapped Phosphorothioate Chimera, e.g.[2′-O-Me]/PS.[2′-OH]/PS Oligoribonucleotide

In the manner of Example 8a chimeric compound having an “open gap”segment at its 3′ terminus,” a “wing” segment at its 5′ terminus andphosphorothioate linkages through out is prepared.

ii. “5′-End” Gapped Phosphorothioate Chimera, e.g.[2′-OH]/PS.[2′-O-Me]/PS Oligoribonucleotide

In the manner of Example 8a chimeric compound having an “open gap”segment at its 5′ terminus,” a “wing” segment at its 3′ terminus andphosphorothioate linkages through out is prepared.

iii. “3′-End” Gapped Phosphorothioate Chimera, e.g. [2′-F]/PS.[2′-OH]/PSOligoribonucleotide

In the manner of Example 8, a chimeric compound having an “open gap” atits 3′ terminus”, 2′-fluoro nucleosides in its 5′ “wing” segment, 2′-OHnucleosides in its open “gap” segment and phosphorothioate linkagesthrough out is prepared.

Example 13 Chimeric Oligoribonucleotides with Uniform Backbone Linkagesand Variable Nucleoside Subunits i. Chimeric 2′-O-ethyloligoribonucleotide, e.g., [2′-O-Et]/PS.[2′-OH]/PS.[2′-O-Et]/PSOligoribonucleotide

In the manner of Example 8a chimeric compound having 2′-O-ethylnucleosides in its “wing” segments, 2′-OH nucleosides in its “gap”segment and phosphorothioate linkages throughout is prepared.

ii. Chimeric 2′-O-propyl Oligoribonucleotide, e.g.,[2′-O-Pr]/PS.[2′-OH]/PS.[2′-O-Pr]/PS Oligoribonucleotide

In the manner of Example 8a chimeric compound having 2′-O-propylnucleosides in its “wing” segments, 2′-OH nucleosides in its “gap”segment and phosphorothioate linkages throughout is prepared.

iii. [2′-O—F]/PS.[2′-OH]/PS.[2′-O—F]/PS Oligoribonucleotide

In the manner of Example 8a chimeric compound having 2′-fluoronucleosides in its “wings” segments, 2′-OH nucleosides in its “gap”segment and phosphorothioate linkages throughout is prepared.

iv. [2′-O-EtOMe]/PS.[2′-OH]/PS.[2′-O-EtOMe]/PS Oligoribonucleotide

In the manner of Example 8a chimeric compound having 2′-O-methoxyethylnucleosides in its “wings” segments, 2′-OH nucleosides in its “gap”segment and phosphorothioate linkages through out is prepared.

v. [2′-O-EtOMe]/PS.[2′-OH]/PS.[2′-F]/PS Oligoribonucleotide

In the manner of Example 8a chimeric compound having 2′-O-methoxyethylnucleosides in its 5′ “wing” segment, 2′-OH nucleosides in its “gap”segment, 2′-fluoro nucleosides in its 3′ “wing” segment, andphosphorothioate linkages through out is prepared.

vi. [2′-O-EtOMe]/PS.[2′-OH]/PS.[2′-O-Me]/PS Oligoribonucleotide

In the manner of Example 8, a chimeric compound having 2′-O-methoxyethylnucleosides in its 5′ “wing” segment, 2′-OH nucleosides in its gap,2′-O-methyl nucleosides in its 3′ “wing” segment and phosphorothioatelinkages through out is prepared.

Example 14 Chimeric Oligoribonucleotides Having Variable BackboneLinkages and Variable Nucleosides i. [2′-O-Me]/PMe.[2′-OH]/PS.[2′-F]/PSOligoribonucleotide

In the manner of Example 8 using chemistries of Example 6, a chimericcompound having 2′-O-methyl nucleosides in its 5′ “wing” segment, 2′-OHnucleosides in its “gap,” 2′-O-fluoro nucleosides in its 3′ “wing”segment, phosphorothioate linkages in the “gap” segment and the 3′“wing” segment and methyl phosphonate linkages in the 5′ “wing” segmentis prepared.

ii. [2′-O-Me]/PME.[2′-OH]/PS.[2′-Pr]/PI Oligoribonucleotide

In the manner of Example 8 using chemistries of Example 6, a chimericcompound having 2′-O-methyl nucleosides in its 5′ “wing” segment, 2′-OHnucleosides in its “gap,” 2′-O-propyl nucleosides in its 3′ “wing”segment, phosphorothioate linkages in the “gap” segment, methylphosphonate linkages in 5′ “wing” segment and phosphinate linkages inthe 3′ “wing” segment is prepared.

Example 15 Chimeric Oligoribonucleotides that Include SurrogateNucleosides i. Morpholino Nucleoside Surrogate ContainingOligoribonucleotide, e.g., [Morpholino NucleosideSurrogate].[2′-OH]/PS.[Morpholino Nucleoside Surrogate]Oligoribonucleotide

In the manner of Examples 7 and 8, a chimeric compound having morpholinonucleosides prepared as per the teachings of U.S. Pat. No. 5,506,337 inits “wing” segments and 2′-OH nucleosides linked via phosphorothioatelinkages in its “gap” segment is prepared.

ii. Cyclobutyl Nucleoside Surrogate Containing Oligoribonucleotide,e.g., [Cyclobutyl Nucleoside Surrogate]/PS.[2′-OH]/PS.[CyclobutylNucleoside Surrogate]/PS Oligoribonucleotide

In the manner of Examples 7 and 8, a chimeric compound having cyclobutylsurrogate nucleosides prepared as per the teachings of U.S. Pat. No.5,359,044 in its “wing” segments, 2′-OH nucleosides in its “gap” segmentand phosphorothioate linkages through out is prepared.

iii. Pyrrolidine Nucleoside Surrogate Containing Oligoribonucleotide,e.g., [Pyrrolidine Nucleoside Surrogate]/PS.[2′-OH]/PS.[PyrrolidineSugar]/PS Oligoribonucleotide

In the manner of Examples 7 and 8, a chimeric compound havingpyrrolidine surrogate nucleosides prepared as per the teachings of U.S.Pat. No. 5,519,135 in its “wing” segments, 2′-OH nucleosides in its“gap” segment and phosphorothioate linkages through out is prepared.

iv. “3′-End” Gapped PNA.Phosphorothioate Chimera, e.g. PNA.[2′-OH]/PSOligoribonucleotide

In the manner of Example 8 in combination with the chemistry of Examples7-b, a chimeric compound having an “open gap” at its 3′ terminus” formedfrom 2′-OH nucleosides having phosphorothioate linkages and PNAsurrogate nucleosides in the 5′ “wing” segment, is prepared.

Example 16 Chimeric Oligoribonucleotides that Include Nucleosides HavingModified Bases i. N-2 Modified Purine Containing Oligoribonucleotide,e.g., [Mod-Purine]/PS.[2′-OH]/PS.[Mod-Purine]/PS Oligoribonucleotide

In the manner of Example 8, a chimeric compound having4,7,10,13-tetraazahexadec-1-yl guanosine nucleosides prepared as per theteachings of U.S. Pat. No. 5,459,255 in its “wing” segments, 2′-OHnucleosides in its “gap” and phosphorothioate linkages through out isprepared.

ii. C-5 Modified Pyrimidine Containing Oligoribonucleotide, e.g.,[Mod-pyr]/PS.[2′-OH]/PS.[Mod-pyr]/PS Oligoribonucleotide

In the manner of Example 8, a chimeric compound having 5-propynylpyrimidine nucleosides prepared as per the teachings of U.S. Pat. No.5,484,908 in its “wing” segments, 2′-OH nucleosides in its “gap” segmentand phosphorothioate linkages through out is prepared.

iii. N-2, C-6 Modified Purine Containing Oligoribonucleotide, e.g.,[Mod-Purine]/PS.[2′-OH]/PS.[Mod-Purine]/PS Oligoribonucleotide

In the manner of Example 8, a chimeric compound having6-hydroxy-2-fluoro purine nucleosides prepared as per the teachings ofU.S. Pat. No. 5,459,255 in its “wing” segments, 2′-OH nucleosides in its“gap” and phosphorothioate linkages through out is prepared.

iv. 2′-O-alkyl, C-5 Modified Pyrimidine Containing Oligoribonucleotide,e.g., [2′-O-Propyl-Mod-pyr]/PS.[2′-OH]/PS.[2′-O-propyl-Mod-pyr]/PSOligoribonucleotide

In the manner of Example 8, a chimeric compound having2′-O-propyl-5-methyl cytidine nucleosides in its “wing” segments, 2′-OHnucleosides in its “gap” segment and phosphorothioate linkages throughout is prepared.

v. 2′-O-alkyl, N-2, C-5 Modified Pyrimidine ContainingOligoribonucleotide, e.g.,[2′-O-propyl-Mod-pyr]/PS.[2′-OH]/PS.[2′-O-propyl-Mod-pyr]/PSOligoribonucleotide

In the manner of Example 8, a chimeric compound having2′-O-propyl-2-thio-5-methyl uridine nucleosides in its “wing” segments,2′-OH nucleosides in its “gap” segment and phosphorothioate linkagesthrough out is prepared.

vi. 2′-O-aminoalkyl, N-2, C-5 Modified Pyrimidine ContainingOligoribonucleotide, e.g.,[2′-β-aminopropyl-Mod-pyr]/PS.[2′-OH]/PS.[2′-β-aminopropyl-Mod-pyr]/PSOligoribonucleotide

In the manner of Example 8, a chimeric compound having2′-O-aminopropyl-2-thio-5-methyl uridine nucleosides in its “wing”segments, 2′-OH nucleosides in its “gap” segment and phosphorothioatelinkages through out is prepared.

vii. 2′-O-fluoro, N-2, C-5 Modified Pyrimidine ContainingOligoribonucleotide, e.g.,[2′-O-fluoro-Mod-pyr]/PS.[2′-OH]/PS.[2′-O-fluoro-Mod-pyr]/PSOligoribonucleotide

In the manner of Example 8, a chimeric compound having2′-O-fluoro-2-thio-5-methyl uridine nucleosides in its “wing” segments,2′-OH nucleosides in its “gap” segment and phosphorothioate linkagesthrough out is prepared.

Example 17 Cell Culture and Northern Blot Analysis of Ras Target

T24 cells were maintained as monolayers in McCoys medium (GIBCO-BRL,Gaithersburg, Md.) supplemented with 10% fetal bovine serum and 100units/ml penicillin. After treatment with oligomeric compounds for 24hrs the cells were trypsinzed, centrifuged and total cellular RNA wasisolated according to standard protocols (see Ausubel et al., CurrentProtocols in Molecular Biology, 1988, Wiley and Sons, New York, N.Y.).To quantify the relative abundance of Ha-ras mRNA, total RNA (10 ug) wastransferred by northern blotting onto Bio-Rad Zeta probe membrane(Bio-Rad, Hercules, Calif.) and UV crosslinked (Stratalinker™,Stratagene, LaJolla, Calif.). Membrane bound RNA was hybridized to a ³²Plabeled 0.9 kb Ha-ras cDNA probe (Oncogene Science, Pasadena, Calif.)and exposure to XAR film (Kodak, Rochester, N.Y.). The relative amountof Ha-ras signal was determined by normalizing the Ha-ras signal to thatobtained when the same membrane was stripped and hybridized with a probefor human glyceraldehyde 3-phosphate dehydrogenase (G3PDH, Clontech,Palo Alto, Calif.). Signals from northern blots were quantified usingphosphoimager and image quant software (Molecular Dynamics, Sunnyvale,Calif.).

Example 18 Compound Treatment of Cells

Cells growing in monolayer were washed once with warm PBS then Opti-MEM(GIBCO-BRL) medium containing Lipofectin (GIBCO-BRL) at a concentrationof 5 ug/ml per 200 nM of oligo with a maximum concentration of 15 ug/mlwas added. Oligomeric compounds were added and incubated at 37° C. for 4hrs when the medium was replaced with full serum medium. After 24 hrs inthe presence of the compound the cells were harvested and RNA preparedfor further analysis.

Example 19 RNase H Analysis

RNase H analysis was performed using 17 base oligoribonucleotidescorresponding to bases (+23 to +47) of activated (codon 12 mutation)Ha-ras mRNA. 5′ End labeled RNA (20 nM) was incubated with a 100-foldmolar excess of the various test oligoribonucleotides in a reactioncontaining 20 mM Tris-Cl, pH 7.5, 100 mM KCl, 10 mM MgCL₂, 1 mMdithiothreitol, and 4 units of RNase inhibitor (Pharmacia, Newark, N.J.)in a final volume of 100 ul. The oligoribonucleotides were melted byheating to 95° C. for 5 minutes then allowed to cool slowly to roomtemperature in 2 liters bath of water 90° C. Duplex formation wasconfirmed by the shift in mobility between the single stranded endlabeled sense RNA and the annealed duplex on non denaturingpolyacrylamide gels. The resulting duplexes were tested as substratesfor digestion by E. coli RNase H (USB, Cleveland, Ohio). 1 μl of a1×10⁻⁹ mg/ml solution of RNase H was added to 10 μl of the duplexreaction incubated at 37° C. for 30 minutes, the reaction was terminatedby the addition of denaturing loading buffer and reaction products wereresolved on a 12% polyacrylamide gel containing 7 M Urea and exposed toXAR film (Kodak).

Example 20 Cell Free In Vitro Nuclease Assay

Duplexes used in the cell free T24 extract experiments were annealed asdescribed above with the exception that after formation of the duplex,the reaction was treated with 1 μl of a mixture RNase T and A (AmbionRPAII kit, Austin, Tex.) and incubated for 15 min at 37° C., and thengel purified from a nondenaturing 12% polyacrylamide gel. T24 cellnuclear and cytosolic fractions were isolated as described previously(Szyf, M., Bozovic, V., and Tanigawa, G., J. Biol. Chem., 1991, 266,10027-10030). Annealed duplexes (10 μl) were incubated with 3 μg of theT24 cytosolic extract at 37° C. The reaction was terminated byphenol/chloroform extraction and ethanol precipitated with the additionof 10 μg of tRNA as a carrier. Pellets were resuspended in 10 μl ofdenaturing loading dye, products were resolved on 12% denaturingacrylamide gels as described above. ³²P-labeled 17-base RNA washydrolysed by heating to 95° C. for 10 minutes in the presence of 50 mMNaCO₃, pH=9.0 to generate a molecular weight ladder.

Example 21 Determination of 5′ and 3′ Termini

Non-labeled duplex was treated with T24 extracts as done previously,half of this reaction was treated with calf intestinal phosphatase (CIP,Stratagene) and half was left untreated. The phosphatase was inactivatedby heating to 95° C. and the reactions were extracted withphenol/chloroform and then precipitated in ethanol with glycogen as acarrier. The precipitates were then treated with T4 polynucleotidekinase (Stratagene) and ³²P-γ-ATP (ICN, Irvine, Calif.). The sampleswere again extracted by phenol/chloroform and precipitated with ethanol,the products of the reaction were then resolved on a 12% acrylamide geland visualized by exposure to XAR film. The 3′-terminus of the cleavedduplex was evaluated by the reaction of duplex digestion products withT4 RNA ligase (Stratagene) and ³²P-pCp (ICN).

Example 22 Chimeric 2′-methoxy Oligoribonucleotides Mediate Digestion ofTarget RNA in T24 Cells

Structure activity analyses of antisense oligonucleotides specific forcodon 12 of the Ha-ras oncogene containing various 2′-sugarmodifications were reported by Monia, et al., J. Biol. Chem., 1992, 267,19954-19962 and Monia et al., J. Biol. Chem., 1993, 268, 14514-14522. Inthose reports, although the 2′-modified oligonucleotides hybridized withgreater affinity to RNA than did unmodified 2′-deoxy oligos they werecompletely ineffective in inhibiting Ha-ras gene expression. The lack ofactivity observed with these 2′-modified oligos was directly attributedto their inability to create duplexes that could serve as substrates fordegradation by RNase H. Following a similar protocol, stretches ofribonucleotides were introduced into the center of 17 base 2′-methoxyoligoribonucleotides targeting Ha-ras mRNA to form2′-methoxy-2′-hydroxy-2′-methoxy phosphorothioate oligoribonucleotide“gapped” chimeric compounds that have varying ribonucleotide content inthe central gap segment (see FIG. 1 for a representation of thesecompounds as well as their base sequence). When hybridized to theircellular target the resultant duplex consists of two stretches that arenot targets for nucleolytic degradation (the 2′-methoxy “wings”) and one2′-hydroxyl oligoribonucleotide stretch that was found to be a targetfor a novel ribonuclease activity that recognizes RNA:RNA duplexes. T24human bladder carcinoma cells were used that contain an activating G-Ttransversion mutation in the Ha-ras gene at the codon 12 position. The“gapped” chimeric compounds specific for this mutation were transfectedinto T24 cells growing in culture. After incubation with the compoundsfor 24 hrs, cells were harvested, total cytosolic RNA isolated andNorthern blot analysis for Ha-ras mRNA levels performed. Fully modified2′-methoxy oligonucleotides did not support nucleolytic cleavage oftarget mRNA and therefore did not lead to a reduction in steady statelevels of Ha-ras mRNA even at the highest concentration tested (FIGS. 2Aand 2B). An RNA gapmer oligonucleotide with only 3 ribonucleotides inthe gap was also incapable of inducing nucleolytic cleavage of thetarget RNA (FIGS. 2C and 2D). However, T24 cells treated with RNA gapmeroligonucleotides containing 5, 7 and 9 ribonucleotides in the gap aswell as a full phosphorothioate oligoribonucleotide molecule alldisplayed dose dependent reductions in Ha-ras steady state mRNA levels(FIGS. 3B-3D). T24 cells treated with a control 9 RNA gapmeroligonucleotide that contained four mismatched bases in its sequence didnot show dose dependent reduction in Ha-ras mRNA suggesting thathybridization to the target RNA is essential for activity (FIG. 3E). TheRNA gapmer compounds showed dose dependent inhibition of Ha-ras steadystate mRNA levels.

The ability of the RNA gapmer compounds to reduce Ha-ras mRNA wasdependent on the size of the RNA gap and thus the size of the RNA:RNAduplex formed in vivo. Treatment of cells with the 3 base RNA gapmercompounds resulted in no cleavage of the target whereas the 5, 7 and 9base RNA gapmer compounds resulted in reduction in Ha-ras mRNA (FIG. 4).The fact that the RNA gapmer oligonucleotide containing 3ribonucleotides in the gap was unable to induce reduction in target mRNAsuggests that the activity involved requires a minimal RNA:RNA duplexregion of at least four ribonucleotides for binding and cleavage of thetarget. Interestingly, chimeric DNA gapmer oligonucleotides that containdeoxynucleotides in the gap instead of ribonucleotides show the sameminimal gap size requirements to form substrates for RNase H mediateddegradation of the target mRNA (Crooke et al., Annu. Rev. Pharmacol.,1996, 36, 107), suggesting that RNase H and the double stranded RNaseactivity described here may share some properties, although theirsubstrates are clearly different.

A control 9 RNA gapmer compound that contains four mismatched bases inits sequence resulted in essentially no reduction in Ha-ras mRNA asexpected as is shown in FIG. 3E. A full phosphorothioateoligoribonucleotide molecule had approximately the same activity as the5 RNA gapmer oligo (FIG. 3D). This might have been due to the relativedecrease in stability of the full oligoribonucleotide in vivo resultingfrom inactivation by single stranded ribonucleases, as 2′-methoxyphosphorothioate oligodeoxynucleotides are considerably more stable thanphosphorothioate oligoribonucleotides. Crooke et al., J. Pharmacol Exp.Ther., 1996, 277, 923-937. Treatment of T24 cells with the variousoligonucleotides and various concentrations up to 800 nM was done intriplicate and quantification of Ha-ras mRNA levels indicate that at 600nM the 5 gapmer reduces Ha-ras mRNA by 51%, the 7 gapmer by 49%, the 9gapmer by 77% and the full ribonucleotide by 38% when compared to nontreated controls. This suggests that RNA gapmer oligoribonucleotidesprotected by 2′-methoxy wings would be more potent molecules. As shownin this example, an endoribonuclease activity in T24 human bladdercarcinoma cells recognizes the internal RNA:oligoribonucleotide portionof a chimeric duplex and reduced the target mRNA levels.

Example 23 An Activity Present in T24 Cellular Extracts Induces Cleavageof Gapmer Oligoribonucleotide:RNA Duplex Within the Internal RNA:RNAPortion In Vitro

To further characterize the double-stranded RNA cleavage activity in T24cells, T24 cellular extracts were prepared and tested for the ability tocleave the 9 gap oligoribonucleotide:RNA duplex in vitro. The 9 gapcompound:³²P-end labeled RNA duplex was incubated with 3 μg of cytosolicextract at 37° C. for varying time periods as shown in FIG. 4, followedby phenol chloroform extraction ethanol precipitation and separation ofthe products on a denaturing gel. That this duplex was a substrate fordigestion by an activity present in T24 extracts is shown by the loss offull length end labeled RNA and the appearance of lower molecular weightdigestion products indicated by arrows in FIG. 4. In addition, theactivity responsible for the cleavage of the duplex has specificity forthe RNA:RNA portion of the duplex molecule, as indicated by the sizes ofthe cleavage products it produces (see the physical map of the ³²P-endlabeled RNA, far right in FIG. 4. RNase H cleavage of a 9deoxynucleotide gap oligonucleotide:RNA duplex and cleavage of the 9ribonucleotide gap oligoribonucleotide:RNA duplex by T24 cellularextracts appears to result in similar digestion products. This is seenby comparing the gels of FIGS. 4 and 5. Both activities displayedpreferred cleavage sites near the 3′ end of the target RNA in theirrespective duplexes which suggests that they may share binding as wellas mechanistic properties. Cellular extracts prepared from humanumbilical vein epithelial cells (HUVEC), human lung carcinoma (A549) andHela cell lines all contained an activity able to induce cleavage of the9 RNA gapmer:RNA target duplex in vitro.

Example 24

Cleavage of Target RNA in Both Cytoplasmic and Nuclear Fractions of CellProducts

The cellular distribution of the double stranded RNase activitydescribed herein was further evaluated. Nuclear extracts were preparedfrom T24 cells and tested for the ability to digest the 9 RNA gapmeroligonucleotide:RNA duplex. Nuclear extracts prepared from T24 cellswere able to degrade the target duplex, and the activity was found to bepresent in the nuclear fraction at comparable levels to that in thecytoplasmic fractions.

An RNA gapmer oligonucleotide was synthesized that containedphosphorothioate linkages throughout the entire length of the molecule.Since this results in increased stability to single stranded nucleases,it was reasoned that it would inhibit cleavage of the antisense strandby the dsRNase as well. Therefore, to determine if the activitydescribed above can cleave both strands in a RNA duplex molecule, a 9RNA gapmer antisense oligonucleotide that contained phosphorothioatelinkages in the wings between the 2′ methoxy nucleotides but hadphosphodiester linkages between the nine ribonucleotides in the gap wassynthesized. A duplex composed of this ³²P-labeled 9 RNA gapmerphosphodiester/phosphorothioate oligonucleotide and its complementaryoligoribonucleotide was tested as a substrate for double stranded RNaseactivity in T24 extracts. The activity was capable of cleaving theantisense strand of this duplex as well as the sense strand and thepattern of the digestion products indicated that cleavage was againrestricted to the RNA:RNA phosphodiester portion of the duplex.

Example 25 An RNA Gapmer Oligonucleotide:RNA Duplex is not a Substratefor RNase H

To exclude the possibility that the cleavage shown in Example 23 mightbe due to RNase H, the ability of E. coli RNase H to cleave a 17 basepair duplex of the 9 gapmer oligoribonucleotide and its complementary 5′³²P-labeled RNA in vitro was tested. FIG. 5 shows the expected shift inelectrophoretic mobility when duplexes were formed and analyzed on anative gel next to the single stranded ³²P-end labeled RNA. As can beseen in FIG. 5 in the far right panel, the 9 Gapmeroligoribonucleotide:RNA duplex was not a substrate for RNase H cleavageas no lower molecular weight bands appeared when it was treated withRNase H. However, as expected a full deoxy oligonucleotide:RNA duplexwas cleaved by RNase H under the same conditions, as is evident by theappearance of lower molecular species in the enzyme treated lane (FIG.5, left panel). A duplex composed of a 9 gapmer DNA oligonucleotide andits complementary RNA was a substrate for RNase H cleavage. The factthat the RNase H cleavage sites in this particular duplex were localizedto the DNA:RNA portions of the duplex further demonstrates that the RNAgapmer oligoribonucleotide:RNA duplex is not a substrate for RNase Hdigestion.

It is interesting to note that RNase H cleavage of the 9 DNA gapmeroligonucleotide:RNA duplex (FIG. 5, left panel) and cleavage of the 9RNA gapmer oligonucleotide:RNA duplex by T24 cellular extracts resultedin similar digestion products (see FIG. 4). Both RNase H and theactivity in T24 cells displayed the same preferred cleavage sites ontheir respective duplexes. Moreover, at this site, both theoligonucleotides were roughly comparable in potency. Cleavage wasrestricted to the 3′ end of the target RNA in the region opposite eitherthe DNA or RNA gap of the respective antisense molecule.

While not wishing to be bound by any particular theory, the immediatelypreceding result suggests that RNase H and the dsRNase of the inventionmay share binding as well as mechanistic properties. However, analysisof DNA and RNA gapmer oligonucleotides targeting four sites on c-RafmRNA revealed that RNase H and the dsRNase activity described hereclearly have different substrate specificities. “RNA-like” gapmeroligonucleotides targeted to the c-Raf mRNA were not able to inducereduction in mRNA whereas RNase H active oligodeoxynucleotides targetedto the same site were able to reduce target mRNA levels. To determine ifthe lack of cleavage induced by the four c-Raf “RNA gapmers” in T24cells was due to possible sequence specificity or cleavage, the fourc-Raf “RNA-like gapmers,” the ras “RNA-like gapmer” and thecorresponding “DNA-like gapmers” were prehybridized to ³²P-labeledtarget oligoribonucleotides and incubated with T24 homogenates. The ras“RNA-like gapmer” supported cleavage of the ras target RNA almost asefficiently as the “DNA gapmer.” However, only one of the “RNA-likegapmers” targeted to c-Raf segments (SEQ ID NO:8) supported any cleavageand the rate of cleavage for the “RNA-like gapmer” was much slower thanthe comparable “DNA-like gapmer.” Thus, in contrast to RNase H, thedsRNase displays considerable sequence specificity.

Example 26 Nuclease Activity Generates 5′-phosphate and 3′-hydroxylTermini

To determine the nature of the 5′ termini left by nuclease cleavage ofthe duplex in vitro, non-labeled duplex was incubated with T24 cellularextracts as previously described then reacted with T4 polynucleotidekinase and [³²P-γ-ATP] with or without prior treatment with calfintestinal phosphatase. Phosphatase treatment of the duplex products wasseen to be essential for the incorporation of ³²P label during thereaction with polynucleotide kinase, indicating the presence of aphosphate group at the 5′ termini. The 3′ termini were evaluated by thereaction of duplex digestion products with T4 RNA ligase and ³²P-pCp. T4RNA ligase requires a free 3′-hydroxyl terminus for the ligation of³²P-pCp. The ability of the duplex digestion products to incorporate³²P-pCp by T4 RNA ligase indicates the presence of 3′-hydroxyl groups.

Example 27 Purification and Characterization of Double-StrandedRibonucleases from Mammalian Tissues

In order to determine if mammalian cells, other than cultured celllines, contain double-strand

RNase activity, and to provide a source from which such ribonucleasesmight be purified, the following efforts were undertaken to identify andpurify dsRNases from rat liver homogenates.

Example 27-a Substrates and Assays for dsRNases

In preliminary experiments, double-strand RNase activity was observed inrat liver homogenates, but the homogenates also displayed high levels ofsingle-strand RNases that complicated analysis of the dsRNase activitiesbecause of cleavage of the oligoribonucleotide overhangs after cleavageby the dsRNases. To solve this problem, two additional substrates and anon-denaturing gel assay were used. The “sense” strand was anoligoribonucleotide having phosphodiester linkages in an eight-base gapwith flanks having either (a) residues with phosphorothioate linkages or(b) 2′-methoxynucleosides with phosphorothioate linkages. The“antisense” strand in both substrates contained 2′-methoxyphosphorothioate wings on either side of an eight-base ribonucleotidegap having either phosphodiester or phosphorothioate linkages (Table 1).Such dsRNase substrates were more stable to exonuclease digestion thanan oligoribonucleotide and substrates with both phosphorothioatelinkages and 2′-methoxy nucleosides was extremely stable. These featuresare important because of the abundance of single-strand RNases relativeto the double-strand RNase activity in the rat liver and supported theuse of non-denaturing assays.

TABLE 1 Artificial Substrates for Mammalian dsRNases* Ha-ras TARGETEDSENSE/ANTISENSE OLIGONUCLEOTIDES SEQ ID NO: 1 5′-GGG CGC CGU CGG UGUGG-3′ SEQ ID NO: 2 3′-CCC GCG GCA GCC ACA CC-5′ C-raf TARGETEDSENSE/ANTISENSE OLGONUCLEOTIDES SEQ ID NO: 3 5′-CCG AAU GUG ACC GCC UCCCG-5′ SEQ ID NO: 4 3′-GGC UUA CAC UGG CGG AGG GC-3′ SEQ ID NO: 5 5′-UCAAUG GAG CAC AUA CAG GG-3′ SEQ ID NO: 6 3′-AGU UAC CUC GUG UAU GUC CC-5′SEQ ID NO: 7 5′-AAU GCA UGU CAC AGG CGG GA-3′ SEQ ID NO: 8 3′-UUA CGUACA GUG UCC GCC CU-5′ *Emboldened residues indicate 2′-methoxynucleotideresidues in the “antisense” strands.

Both rat liver cytosolic and nuclear extracts induced cleavage of theduplex substrate. Both extracts resulted in more rapidly migrating bandson native gel electrophoretic analyses. The cytosolic extract appearedto be more active than the nuclear extract. A double-strand RNase, RNaseV1 (Pharmacia, Piscataway, N.J.) cleaved the substrate; T24 extractsalso cleaved the substrate. Neither bacterial nor single-strand RNasecleaved the substrate, with the exception of RNase A, which at very highconcentrations resulted in some cleavage. It is unclear whether thatcleavage was due to a contaminating double-strand RNase or if RNase Acan, under some conditions, cleave double-strand substrates.

Example 27-b Purification of dsRNases from rat Liver Cytosolic andNuclear Extracts

In order to purify the mammalian dsRNase identified herein, 0.5 kg ofrat liver was homogenized in Buffer×[10 mM Hepes (ph 7.5), 25 mM KCl,0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M sucrose, 10%glycerol; all reagents from Sigma Chemical Co., St. Louis, Mo.] andcentrifuged in a Beckman J2-21M centrifuge (Beckman, Fullerton, Calif.)at 10,000 rpm for 1.5 hours. The supernatant was precipitated with 40%ammonium sulfate (Sigma). All the dsRNase activity was recovered in the40% ammonium sulfate precipitate. The pellet was resuspended in Buffer A[20 mM Hepes (ph 6.5), 5 mM EDTA, 1 mM DTT, 0.25 mM phenylmethylsulfonylfluoride (PMSF), 0.1 M KCl, 5% glycerol, 0.1% NP40, 0.1% Triton X-100;all reagents from Sigma] and dialyzed to remove ammonium sulfate.Approximately 40 g of cytosolic extract were obtained from 0.5 kg liver.

A crude nuclear pellet, prepared as in the previous Examples, wasresuspended and homogenized in Buffer Y [20 mM Hepes (ph 7.5), 0.42 MNaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 25% glycerol].The homogenate was centrifuged in a J2-21M centrifuge (Beckman) at10,000 rpm for 1.5 hrs. The supernatant was precipitated with 70%ammonium sulfate. The pellet was resuspended in Buffer A and dialyzed.All the dsRNase activity was recovered in the 70% ammonium sulfateprecipitate. Approximately 5 g Of nuclear extract were obtained from 0.5kg liver.

Ion exchange chromatography was then performed in order to furtherpurify the dsRNases of the invention. Nuclear and cytosolic extracts inBuffer A were loaded onto Hi-Trap columns (Pharmacia, Piscataway, N.J.)for FPLC. The extracts were eluted with a linear gradient of NaCl andsamples were collected. The UV absorption at 257 ηM of the samples wasdetermined. Samples were centrifuged at 8,000 g for 10 minutes,resuspended in Buffer A, concentrated in Ultrafree-15 centrifugal filterdevices (Millipore, Bedford, Mass.) and analyzed for activity. ThedsRNase activity eluted in fractions corresponding 300-450 mM NaCl. Incontrast, the dsRNase activity in the nuclear extract eluted at 700-800mM NaCl.

Fractions from the ion exchange chromatography were concentrated andsubjected to size exclusion chromatography. Active samples from the ionexchange chromatography were pooled, applied to a TSK G-3000 column(TosoHaas, Mongomeryville, Pa.) and run with Buffer A containing 100 mMNaCl. Samples (200 to 400 ul) were collected and their UV absorption at257 ηM was determined. Samples were concentrated using Ultrafree-15centrifugal filter devices (Millipore) and then analyzed for activity.

FIG. 6 shows a polyacrylamide gel electrophoretic analysis of theconcentrated active fractions after the ion-exchange chromatography, andthe fractions from the size exclusion chromatography. The fraction withgreatest dsRNase activity (lane 4, FIG. 6) had a molecular weight rangeof about 50 to about 80 kilodaltons, and a band at approximately 50kilodaltons appeared to be enhanced on 12% polyacrylamide gelelectrophoresis (PAGE) performed using precast gels (Novex, San Diego,Calif.).

Table 2 provides a summary of the purification and recovery of dsRNaseactivities from nuclear and cytosolic liver extracts.

TABLE 2 Summary of Purification of dsRNases from Rat Liver HomogenatesTotal Specific Protein Activity Activity Purification Recovery Fraction(mg) (units*) (unit/mg) Factor (%) Cytosolic 30,000 1,020,000 34 1 100extract Ion 991 459,000 463 14 56 Exchange (Pool) Gel 18.4 100,980 5,600165 22 FiltrationOne unit is defined as the amount of sample required to digest 10 fMoldsRNA duplex in 15 minutes at 37° C. under the conditions describedherein.

Purification of the dsRNase activities from liver nuclei and cytosolsuggests that at least two dsRNases with differing properties arecapable of cleaving double-strand RNA. The nuclear dsRNase eluted athigher NaCl concentrations from the ion exchange column than thecytosolic dsRNase. However, both require Mg⁺⁺ and cleave at severalsites within the oligoribonucleotide gap. Both require a duplexsubstrate and can cleave oligoribonucleotides in a duplex that is madeup of oligoribonucleotide “sense” and a 2′ methoxy phosphorothioatechimeric “antisense” strand when the duplex has phosphorothioate orphosphorothioate-2′ methoxy nucleoside wings.

Having (1) established a reproducible and activity-specific assay for,(2) determined several sources of and (3) achieved an adequate degree ofpurification of the dsRNases of the invention via the methods describedabove, the dsRNases are further purified by a variety of means. In allinstances, the use of organic solvents is avoided as the dsRNases of theinvention are unstable in acetonitrile or methanol (see below), and theassays described herein are used to evaluate the presence or absence ofthe desired dsRNase in a sample. Further purification steps may include,but are not limited to, the following means.

Several types of heparin columns have been used to purify a variety ofribonucleases. For example, Sepharose columns have been utilized in thepurification of a sequence-specific ribonuclease from Rana catesbeiana(bullfrog) oocytes (Liao, Nucl. Acids Res., 1992, 20, 1371), aribonuclease from Xenopus laevis oocytes (Nitta et al., Biol. Pharm.Bull. (Jpn.), 1993, 16, 353), several ribonucleases from thethermophilic archaebacterium Sulfobus Solfataricus (Fusi et al., Eur. J.Biochem., 1993, 211, 305), and a ribonuclease from human spleen (Yasudaet al., Eur. J. Biochem., 1990, 191, 523).

Hydrophobic interaction chromatography is a powerful proteinpurification means which depends on strong salting-out salts to increasethe hydrophobic interactions between the desired protein and a ligandtherefor (Narhi et al., Anal. Biochem., 1989, 182, 266). Hydrophobicinteraction columns (HICs) have been used to purify ribonuclease A fromundesired contaminants (Wu et al., Methods in Enzymology, 1996, 270, 27;Wetlaufer et al., J. Chromatography, 1986, 359, 55).

The dsRNases of the invention may also be further purified byhydroxyapatite chromatography (Kennedy, Methods in Enzymology, 1990,182, 339). Endo- and exo-ribonuclease have been purified fromTrypanosoma brucei using hydroxyapatite chromatography (Gbenle, Exp.Parisitol., 1990, 71, 432; Gbenle, Mol. Biochem. Parasitol., 1985, 15,37).

RNA affinity columns may also be used to further purify the dsRNases ofthe invention. In particular, a commercially available double-strandedRNA affinity column (Pharmacia, Piscataway, N.J.) may be used.Alternatively, a column is prepared in which the matrix thereofcomprises one or more of the dsRNase substrates of the invention (fordetails, see the following Example). Due to the relative sequencespecificity of the dsRNase of the present invention, the latter type ofaffinity column may be preferable. In order to prevent degradation ofthe matrix of either type of double-stranded affinity matrix, samplescomprising the dsRNases of the invention are treated in such a manner soas to limit the degradative capacity of the dsRNase withoutsignificantly altering its ability to bind to the double-stranded RNAsubstrate of the matrix. For example, the degradative activity of thedsRNase of the invention is inhibited in solutions lacking availableMg⁺⁺ due to, for example, the addition of appropriate chelating agentssuch as EDTA, or by addition of NaCl to a sample containing suchdsRNases to a final concentration of at least 300 mM (see the followingsubsection).

Those skilled in the art will recognize that the above means, as well asothers not herein described, will need to be optimized for optimalefficiency in purifying the dsRNases of the invention. For example, theselection of one or more of the above means as a further purificationstep, and of the order in which such means are applied, will effect thedegree of purity and specific activity of the dsRNase so treated.However, such optimization is believed to be within the skill of the artgiven that the assays described herein can be readily utilized by askilled artisan to determine the effect of further purification steps onthe activity of the desired dsRNase. Other techniques known in the art,such as SDS-PAGE, can be used to determine the purity of samplessubjected to the above purification means.

Example 27-c Characterization of Purified Mammalian dsRNases

The effects of various conditions on the dsRNase activity were evaluatedusing the active fractions after ion exchange chromatography. ThedsRNase activity was demonstrable in Tris or phosphate buffers fromabout pH 7 to about pH 10. The dsRNase activity was not stable insolution in acetonitrile or methanol. Furthermore, the activity wasinhibited by NaCl; dsRNase activity was inhibited by 30% at 10 mMNaCl, >60% at 100 mM NaCl and 100% at 300 mM NaCl. Heating for fiveminutes at 60° C., 80° C. or 100° C., inactivated the dsRNase. Optimumactivity was seen in the temperature range of about 37° C. to about 42°C. At 25° C., the dsRNase activity was approximately 50% of thatobserved at 37° C. The dsRNase activity was inhibited at 10, 20 and 50mM EDTA, but not at 5 mM, in agreement with its requirement for Mg⁺⁺,and was stable to multiple freeze/thaws.

Example 28 Further Characterization of the dsRNase Cleavage Site UsingPurified Rat dsRNase

The purified dsRNases were used to characterize the site of cleavage inmore detail. Because it was necessary to minimize any single-strandcleavage from occurring after endonuclease cleavage and during handling,particularly after denaturing of the duplex. Consequently, the moststable duplex substrate, i.e., one in which both strands of the duplexcontained flanking regions comprised of 2′ methoxy nucleosides andphosphorothioate linkages was used.

Example 28-a ³²P Labeling of Oligonucleotides

The sense oligonucleotide was 5′-end labeled with ³²P using [g³²P]ATP,T4 polynucleotide kinase, and standard procedures (Ausubel et al.,1989). The labeled oligonucleotide was purified by electrophoresis on12% denaturing PAGE (Sambrook et al., 1989). The specific activity ofthe labeled oligonucleotide was approximately 5000 cpm/fmol.

Example 28-b Double-Strand RNA Digestion Assay

Oligonucleotide duplexes were prepared in 30 uL reaction buffer [20 mMtris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl₂, 0.1 mM DTT] containing 10 nMantisense oligonucleotide and 10⁵ cpm ³²P labeled sense oligonucleotide.Reactions were heated at 90° C. for 5 min and incubated at 37° C. for 2h. The oligonucleotide duplexes were incubated in either unpurified andsemipurified cellular extracts at a total protein concentration of 75 ugunpurified cytosolic extract, 60 ug unpurified nuclear extract, 5 ug ionexchange purified cytosolic fraction, 5 ug ion exchange purified nuclearfraction, or 0.5 ug ion exchange and gel filtration purified nuclearfraction. Digestion reactions were incubated at 37° C. for 0-240 min.Following incubation, 10 uL of each reaction was removed and quenched byaddition of denaturing gel loading buffer [5 uL 8 M urea, 0.25% xylenecyanole FF, 0.25% bromphenol blue]. The reactions were heated at 95° C.for 5 min and resolved in a 12% denaturing polyacrylamide gel. Theremaining aliquot was quenched in 2 uL native gel loading buffer[glycerol, 0.25% xylene cyanole FF. The reactions were resolved at 10°C. in a 12% native polyacrylamide gel containing 44 mM Tris-borate and 1mM MgCl₂. Gels were analyzed using a Molecular Dynamics Phosphorimager.

FIG. 7 displays the native gel results. Lane 1 shows the position atwhich the untreated ³²P-labeled sense strand migrated in the native gel,and lane 2 shows “sense” strand RNA treated with 0.02 units RNase V1. Inthe remaining lanes, the results of treatment of dsRNAse substrates with0.02 (lane 3) and 0.002 (lane 4) units of RNase V1, unpurified nuclearextract for 0 minutes (lane 5) or 240 minutes (lane 6), unpurifiednuclear extract for 240 minutes without Mg⁺⁺ (lane 7), unpurifiedcytosolic extract for 240 minutes (lane 8), ion exchange purifiedcytosolic extract for 240 minutes in the presence (lane 9) or absence(lane 10) of Mg⁺⁺, and ion exchange/gel filtration purified cytosolicextract for 240 minutes in the presence (lane 9) or absence (lane 10) ofMg⁺⁺ are shown.

FIG. 8 shows the results of analysis of products of digestion of dsRNAsesubstrates by denaturing polyacrylamide gel electrophoresis. Lane 1shows “sense” strand RNA treated with 5×10⁻³ units of RNase A, and lane2 shows “sense” strand RNA treated with 0.02 units RNase V1. Theremaining lanes show dsRNAse products treated with 0.02 (lane 3) and0.002 (lane 4) units of RNase V1, with unpurified nuclear extract for 0minutes (lane 5) or 240 minutes (lane 6), with unpurified cytosolicextract for 240 minutes (lane 7), with ion exchange purified cytosolicextract for 240 minutes (lane 8), and with ion exchange/gel filtrationpurified cytosolic extract for 240 minutes (lane 9). Lane 10 is an RNAbase hydrolysis ladder included for sizing purpose. RNase V1 digestionof the single-strand substrate resulted in little degradation (lane 2).RNase V1 digestion of the duplex resulted in degradates reflectingcleavage at several sites within the gap (lanes 3 and 4). In lanes 4-9,the band at the top of the gel demonstrates that even afterdenaturation, some of the duplex remained annealed, reflecting the veryhigh affinity of duplexes comprised to 2′-methoxy nucleosides. Lanes 6-9show that both the nuclear and cytosolic ribonucleases cleaved thetriplex substrate at several sites with the oligoribonucleotide gap andthat the sites of degradation were different from those of RNase V1. Theposition of the degradates in lanes 6-9 is consistent with them beingthe 2′ methoxy phosphorothioate flanking regions (wings).

Example 29 RNA affinity Columns and Methods of Purifying Ribonucleases

Techniques for preparing nucleic acid affinity columns are known in theart (see, e.g., Kadonaga, Methods in Enzymology, 1991, 208, 10). Suchaffinity columns comprise a matrix comprising a nucleic acid substratefor a desired compound that binds the substrate either nonspecificallyor in a sequence-specific manner. Initially utilized in the purificationof DNA-binding proteins, RNA affinity columns have also been employed topurify RNA-binding proteins and ribonucleases (see, e.g., Prokipcak etal., J. Biol. Chem., 1994, 269, 9261; Dake et al., J. Biol. Chem., 1988,263, 7691). A matrix comprising one or more dsRNase substrates of theinvention has the advantage of providing a dsRNA substrate that isresistant to the action of single-stranded ribonuclease which areprevalent in many tissues and cells. Such a matrix also comprises asuitable solid support and a linker that provides a bridge between thesolid support and the dsRNase substrate(s).

Suitable solid supports include, but are not limited to, graft polymers(U.S. Pat. No. 4,908,405 to Bayer and Rapp); polyacrylamide (Fahy etal., Nucl. Acids Res., 1993, 21, 1819); polyacrylmorpholide, polystyreneand derivatized polystyrene resins (Syvanen et al., Nucl. Acids Res.,1988, 16, 11327; U.S. Pat. Nos. 4,373,071 and 4,401,796 to Itakura),including amino methyl styrene resins (U.S. Pat. No. 4,507,433 to Millerand Ts'O); copolymers of N-vinylpyrrolidone and vinylacetate (Selingeret al., Tetrahedron Letts., 1973, 31, 2911; Selinger et al., DieMakromolekulare Chemie, 1975, 176, 609; and Selinger, DieMakromolekulare Chemie, 1975, 176, 1611); TEFLON™ (Lohrmann et al., DNA,1984, 3, 122; Duncan et al., Anal. Biochem., 1988, 169, 104); controlledpore glass (Chow et al., Anal. Biochem., 1988, 175, 63); polysaccharidesupports such as agarose (Kadonaga, Methods Enzymol., 1991, 208, 10;Arndt-Jovin et al., Eur. J. Biochem., 1975, 54, 411; Wu et al., Science,1987, 238, 1247; Blank et al., Nucleic Acids Res., 1988, 16, 10283) orcellulose (Goldkorn et al., Nucl. Acids Res., 1986, 14, 9171; Alberts etal., Meth. Enzymol., 1971, 21, 198) or derivatives thereof, e.g.,DEAE-cellulose (Schott, J. Chromatogr., 1975, 115, 461) orphosphocellulose (Siddell, Eur. J. Biochem., 1978, 92, 621; Bunemann etal., Nucl. Acids Res., 1982, 10, 7163; Noyes et al., Cell, 1975, 5, 301;Bunemann et al., Nucl. Acids Res., 1982, 10, 7181); dextran sulfate(Gingeras et al., Nucl. Acids Res., 1987, 15, 5373); polypropylene(Matson et al., Anal. Biochem., 1994, 217, 306); agarose beads (Kadonagaet al., Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 5889); latex particles(Kawaguchi et al., Nucleic Acids Res., 1989, 17, 6229); nylon beads (VanNess et al., Nucl. Acids Res., 1991, 19, 3345); paramagnetic beads(Gabrielson et al., Nucl. Acids Res., 1989, 17, 6253; Lund, et al.,Nucl. Acids Res., 1988, 16, 10861; Day et al., Biochem. J., 1991, 278,735); silica gels (Yashima et al., J. Chromatogr., 1992, 603, 111);derivatized forms of silica gels, polytetrafluoroethylene, cellulose ormetallic oxides (U.S. Pat. No. 4,812,512 to Buendia); and art-recognizedequivalents of any of the preceding solid supports; microtiter plates(Drmanac et al., Science, 1993, 260, 1649); crosslinked copolymers ofN-vinylpyrrolidone, other N-vinyl-lactam monomers and an ethylenicallyunsaturated monomer having at least one amine or amine-displacablefunctionality as disclosed in U.S. Pat. No. 5,391,667. In one set ofpreferred embodiments, polystyrene or long chain alkyl CPG (controlledpore glass) beads are employed. In another set of preferred embodiments,microscopic glass slides are employed (Fodor et al., Science, 1991, 251,767; Maskos et al., Nucleic Acids Research, 1992, 20, 1679; Guo et al.,1994, 22, 5456; Pease et al., Proc. Natl. Acad. Sci. U.S.A., 1994, 91,5022).

With regard to the linker, a variety of chemical linking groups orchains may be employed in the matrices of the invention. Any chemicalgroup or chain capable of forming a chemical linkage between the solidsupport and the dsRNase substrate may be employed. A suitable linker hasthe preferred characteristic of non-reactivity with compounds introducedduring the various steps of oligonucleotide synthesis. It will beappreciated by those skilled in the art that the chemical composition ofthe solid support and the dsRNase substrate will influence the choice ofthe linker. Many suitable linkers will comprise a primary amine group ateither or both termini, as many chemical reactions are known in the artfor linking primary amine groups to a variety of other chemical groups;however, other terminal reactive moieties are known and may be used inthe invention. Suitable linkers include, but are not limited to, linkershaving a terminal thiol group for introducing a disulfide linkages tothe solid support (Day et al., Biochem. J., 1991, 278, 735; Zuckermannet al., Nucl. Acids Res., 15, 5305); linkers having a terminalbromoacetyl group for introducing a thiol-bromoacetyl linkage to thesolid support (Fahy et al., Nucl. Acids Res., 1993, 21, 1819); linkershaving a terminal amino group which can be reacted with an activated 5′phosphate of an oligonucleotide (Takeda et al., Tetrahedron Letts.,1983, 24, 245; Smith et al., Nucl. Acids Res., 1985, 13, 2399; Zarytovaet al., Anal. Biochem., 1990, 188, 214); poly(ethyleneimine) (Van Nesset al., Nucl. Acids Res., 1991, 19, 3345); acyl chains (Akashi et al.,Chem. Lett., 1988, 1093; Yashima et al., J. Chromatogr., 1992, 603,111); polyvinyl alcohol (Schott, J. Chromatogr., 1975, 115, 461); alkylchains (Goss et al., J. Chromatogr., 1990, 508, 279); alkylamine chains(Pon et al., BioTechniques, 1988, 6, 768); biotin-avidin orbiotin-streptavidin linkages (Kasher et al., Mol. Cell. Biol., 1986, 6,3117; Chodosh et al., Mol. Cell. Biol., 1986, 6, 4723; Fishell et al.,Methods Enzymol., 1990, 184, 328); and art-recognized equivalents of anyof the preceding linkers. In a preferred embodiment of the invention, ann-aminoalkyl chain is the linker. Methods of determining an appropriate(i.e., providing the optimal degree and specificity of hybridizationbetween the sensor array and the target oligonucleotide) linker lengthare known in the art (see, e.g., Day et al., Biochem. J., 1991, 278,735).

1. A composition comprising a duplex consisting of a first chemicallysynthesized oligomeric compound and a second chemically synthesizedoligomeric compound, wherein: each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound independently consists of 17 to 25 linked nucleosides; at least17 contiguous nucleobases of the first chemically synthesized oligomericcompound are 100% complementary to at least 17 contiguous nucleobases ofthe second chemically synthesized oligomeric compound and to a targetmessenger RNA; at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises a plurality of nucleosides comprising a 2′-hydroxylpentofuranosyl sugar moiety; at least one of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises at least one nucleoside comprising a2′-fluoro sugar modification; and the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound are not covalently linked to each other.
 2. The composition ofclaim 1 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises a central gap region flanked by at least two wingregions.
 3. The composition of claim 1 wherein each of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound comprises a central gap region flankedby at least two wing regions.
 4. The composition of claim 1 wherein atleast one of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises at least4 contiguous nucleosides that comprise 2′-hydroxyl pentofuranosyl sugarmoieties.
 5. (canceled)
 6. (canceled)
 7. (canceled)
 8. The compositionof claim 1 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one nucleoside comprising a 2′ sugarmodification selected from, alkoxy, amino-alkoxy, allyloxy,imidazolylalkoxy, polyethylene glycol, and methoxyethoxy.
 9. Thecomposition of claim 1 wherein each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound independently comprises at least one nucleoside comprising a 2′sugar modification.
 10. The composition of claim 9 wherein at least oneof the 2′ sugar modifications is selected from, alkoxy, amino-alkoxy,allyloxy, imidazolylalkoxy, polyethylene glycol, and methoxyethoxy. 11.The compound of claim 1 wherein at least one of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises a plurality of nucleoside subunitscomprising a 2′-hydroxyl pentofuranosyl sugar moiety and at least onenucleoside comprising a 2′ sugar modification.
 12. The compound of claim1 wherein each of the first chemically synthesized oligomeric compoundand the second chemically synthesized oligomeric compound comprises aplurality of nucleoside subunits comprising a 2′-hydroxyl pentofuranosylsugar moiety and at least one nucleoside comprising a 2′ sugarmodification.
 13. (canceled)
 14. (canceled)
 15. The composition of claim1 wherein at least one of the first chemically synthesized oligomericcompound and the second chemically synthesized oligomeric compoundcomprises at least two nucleosides each comprising a sugar comprising a2′-fluoro.
 16. The composition of claim 1 wherein each of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound comprises at least two nucleosides eachcomprising a sugar comprising a 2′-fluoro.
 17. The composition of claim1 wherein at least one of the first chemically synthesized oligomericcompound and the second chemically synthesized oligomeric compoundcomprises at least one nucleoside comprising a sugar comprising a2′-fluoro and at least one nucleoside comprising a sugar comprising a2′-O-alkyl.
 18. The composition of claim 1 wherein each of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound comprises at least one nucleosidecomprising a sugar comprising a 2′-fluoro and at least one nucleosidecomprising a sugar comprising a 2′-O-alkyl.
 19. The composition of claim1 wherein at least one of the first chemically synthesized oligomericcompound and the second chemically synthesized oligomeric compoundcomprises at least two nucleosides each comprising a sugar comprising a2′-OCH₃.
 20. The composition of claim 19 wherein the 5′ terminalnucleoside of at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises a sugar comprising a 2′-OCH₃.
 21. The composition ofclaim 1 wherein each of the first chemically synthesized oligomericcompound and the second chemically synthesized oligomeric compoundcomprises at least two nucleosides each comprising a sugar comprising a2′-OCH₃.
 22. The composition of claim 21 wherein the 5′ terminalnucleoside of at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises a sugar comprising a 2′-OCH₃.
 23. The composition ofclaim 1 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least two nucleosides comprising different 2′sugar modifications.
 24. The composition of claim 1 wherein the firstchemically synthesized oligomeric compound comprises at least onenucleoside comprising a 2′ sugar modification and the second chemicallysynthesized oligomeric compound comprises at least one nucleoside thatcomprises a different 2′ sugar modification.
 25. The composition ofclaim 1 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one nucleoside comprising a sugar surrogate.26. The composition of claim 1 wherein each of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises at least one nucleoside comprising a sugarsurrogate.
 27. The composition of claim 1 wherein at least one of thefirst chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least onechemically modified internucleoside linkage.
 28. The composition ofclaim 27 wherein at least one chemically modified internucleosidelinkage is a phosphorothioate linkage.
 29. The composition of claim 1wherein each of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises at leastone chemically modified internucleoside linkage.
 30. The composition ofclaim 29 wherein at least one chemically modified internucleosidelinkage is a phosphorothioate linkage.
 31. A composition comprising aduplex consisting of a first chemically synthesized oligomeric compoundand a second chemically synthesized oligomeric compound, wherein: eachof the first chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound independently consists of 17to 25 linked nucleoside subunits; at least 17 contiguous nucleobases ofthe first chemically synthesized oligomeric compound are 100%complementary to at least 17 contiguous nucleobases of the secondchemically synthesized oligomeric compound and to a target messengerRNA; each of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises aplurality of nucleoside subunits comprising a 2′-hydroxyl pentofuranosylsugar moiety; at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one nucleoside comprising a 2′-fluoro sugarmodification; and the first chemically synthesized oligomeric compoundand the second chemically synthesized oligomeric compound are notcovalently linked to each other.
 32. The composition of claim 31 whereinat least one of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises acentral gap region flanked by at least two wing regions.
 33. Thecomposition of claim 31 wherein each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises a central gap region flanked by at least two wingregions.
 34. The composition of claim 31 wherein at least one of thefirst chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least 4contiguous nucleosides that comprise 2′-hydroxyl pentofuranosyl sugarmoieties.
 35. The composition of claim 31 wherein each of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound comprises at least 4 contiguousnucleosides that comprise 2′-hydroxyl pentofuranosyl sugar moieties. 36.The composition of claim 35 wherein the at least 4 contiguousnucleosides comprising 2′-hydroxyl pentofuranosyl sugar moieties of thefirst chemically synthesized oligomeric compound and the at least 4contiguous nucleosides comprising 2′-hydroxyl pentofuranosyl sugarmoieties of the second chemically synthesized oligomeric compoundhybridize to each other in the duplex.
 37. (canceled)
 38. Thecomposition of claim 31 wherein at least one of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises at least one nucleoside comprising a 2′sugar modification selected from, alkoxy, amino-alkoxy, allyloxy,imidazolylalkoxy, polyethylene glycol, and methoxyethoxy.
 39. Thecomposition of claim 31 wherein each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound independently comprises at least one nucleoside comprising a 2′sugar modification.
 40. The composition of claim 39 wherein at least oneof the 2′ sugar modifications is selected from, alkoxy, amino-alkoxy,allyloxy, imidazolylalkoxy, polyethylene glycol, and methoxyethoxy. 41.(canceled)
 42. (canceled)
 43. The composition of claim 31 wherein atleast one of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises at leasttwo nucleosides each comprising a sugar comprising a 2′-fluoro.
 44. Thecomposition of claim 31 wherein each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least two nucleosides each comprising a sugarcomprising a 2′-fluoro.
 45. The composition of claim 31 wherein at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises at least onenucleoside comprising a sugar comprising a 2′-fluoro and at least onenucleoside comprising a sugar comprising a 2′-O-alkyl.
 46. Thecomposition of claim 31 wherein each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one nucleoside comprising a sugar comprisinga 2′-fluoro and at least one nucleoside comprising a sugar comprising a2′-O-alkyl.
 47. The composition of claim 31 wherein at least one of thefirst chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least twonucleosides each comprising a sugar comprising a 2′-OCH₃.
 48. Thecomposition of claim 47 wherein the 5′ terminal nucleoside of at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises a sugarcomprising a 2′-OCH₃.
 49. The composition of claim 31 wherein each ofthe first chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least twonucleosides each comprising a sugar comprising a 2′-OCH₃.
 50. Thecomposition of claim 49 wherein the 5′ terminal nucleoside of at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises a sugarcomprising a 2′-OCH₃.
 51. The composition of claim 31 wherein at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises at least twonucleosides comprising different 2′ sugar modifications.
 52. Thecomposition of claim 31 wherein the first chemically synthesizedoligomeric compound comprises at least one nucleoside comprising a 2′sugar modification and the second chemically synthesized oligomericcompound comprises at least one nucleoside that comprises a different 2′sugar modification.
 53. The composition of claim 31 wherein at least oneof the first chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least onenucleoside comprising a sugar surrogate.
 54. The composition of claim 31wherein each of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises at leastone nucleoside comprising a sugar surrogate.
 55. The composition ofclaim 31 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one chemically modified internucleosidelinkage.
 56. The composition of claim 31 wherein at least one chemicallymodified internucleoside linkage is a phosphorothioate linkage.
 57. Thecomposition of claim 31 wherein each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one chemically modified internucleosidelinkage.
 58. The composition of claim 31 wherein at least one chemicallymodified internucleoside linkage is a phosphorothioate linkage.
 59. Acomposition comprising a duplex consisting of a first chemicallysynthesized oligomeric compound and a second chemically synthesizedoligomeric compound, wherein: each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound independently consists of 17 to 25 linked nucleoside subunits;at least 17 contiguous nucleobases of the first chemically synthesizedoligomeric compound are 100% complementary to at least 17 contiguousnucleobases of the second chemically synthesized oligomeric compound andto a target messenger RNA; at least one of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises a plurality of nucleoside subunitscomprising a 2′-hydroxyl pentofuranosyl sugar moiety; each of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound independently comprises at least onenucleoside comprising a 2′-fluoro sugar modification; and the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound are not covalently linked to each other.60. The composition of claim 59 wherein at least one of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound comprises a central gap region flankedby at least two wing regions.
 61. The composition of claim 59 whereineach of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises a centralgap region flanked by at least two wing regions.
 62. The composition ofclaim 59 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least 4 contiguous nucleosides that comprise2′-hydroxyl pentofuranosyl sugar moieties.
 63. (canceled)
 64. (canceled)65. (canceled)
 66. The composition of claim 59 wherein at least one ofthe first chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least onenucleoside comprising a 2′ sugar modification selected from, alkoxy,amino-alkoxy, allyloxy, imidazolylalkoxy, polyethylene glycol, andmethoxyethoxy
 67. (canceled)
 68. The composition of claim 59 whereineach of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises at least onenucleoside comprising a 2′ sugar modification selected from, alkoxy,amino-alkoxy, allyloxy, imidazolylalkoxy, polyethylene glycol, andmethoxyethoxy.
 69. The compound of claim 59 wherein at least one of thefirst chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises a plurality ofnucleoside subunits comprising a 2′-hydroxyl pentofuranosyl sugar moietyand at least one nucleoside comprising a 2′ sugar modification. 70.(canceled)
 71. (canceled)
 72. (canceled)
 73. (canceled)
 74. (canceled)75. (canceled)
 76. (canceled)
 77. The composition of claim 59 wherein atleast one of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises at leasttwo nucleosides each comprising a sugar comprising a 2′-OCH₃.
 78. Thecomposition of claim 77 wherein the 5′ terminal nucleoside of at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises a sugarcomprising a 2′-OCH₃.
 79. The composition of claim 59 wherein each ofthe first chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least twonucleosides each comprising a sugar comprising a 2′-OCH₃.
 80. Thecomposition of claim 79 wherein the 5′ terminal nucleoside of at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises a sugarcomprising a 2′-OCH₃.
 81. The composition of claim 59 wherein at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises at least twonucleosides comprising different 2′ sugar modifications.
 82. Thecomposition of claim 59 wherein the first chemically synthesizedoligomeric compound comprises at least one nucleoside comprising a 2′sugar modification and the second chemically synthesized oligomericcompound comprises at least one nucleoside that comprises a different 2′sugar modification.
 83. The composition of claim 59 wherein at least oneof the first chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least onenucleoside comprising a sugar surrogate.
 84. The composition of claim 59wherein each of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises at leastone nucleoside comprising a sugar surrogate.
 85. The composition ofclaim 59 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one chemically modified internucleosidelinkage.
 86. The composition of claim 85 wherein at least one chemicallymodified internucleoside linkage is a phosphorothioate linkage.
 87. Thecomposition of claim 59 wherein each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one chemically modified internucleosidelinkage.
 88. The composition of claim 87 wherein at least one chemicallymodified internucleoside linkage is a phosphorothioate linkage.
 89. Acomposition comprising a duplex consisting of a first chemicallysynthesized oligomeric compound and a second chemically synthesizedoligomeric compound, wherein: each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound independently consists of 17 to 25 linked nucleoside subunits;at least 17 contiguous nucleobases of the first chemically synthesizedoligomeric compound are 100% complementary to at least 17 contiguousnucleobases of the second chemically synthesized oligomeric compound andto a target messenger RNA; each of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises a plurality of nucleoside subunits comprising a2′-hydroxyl pentofuranosyl sugar moiety; each of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound independently comprises at least one nucleosidecomprising a 2′-fluoro sugar modification; and the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound are not covalently linked to each other.
 90. Thecomposition of claim 89 wherein at least one of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises a central gap region flanked by at leasttwo wing regions.
 91. The composition of claim 89 wherein each of thefirst chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises a central gapregion flanked by at least two wing regions.
 92. The composition ofclaim 89 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least 4 contiguous nucleosides that comprise2′-hydroxyl pentofuranosyl sugar moieties.
 93. The composition of claim89 wherein each of the first chemically synthesized oligomeric compoundand the second chemically synthesized oligomeric compound comprises atleast 4 contiguous nucleosides that comprise 2′-hydroxyl pentofuranosylsugar moieties.
 94. The composition of claim 93 wherein the at least 4contiguous nucleosides comprising 2′-hydroxyl pentofuranosyl sugarmoieties of the first chemically synthesized oligomeric compound and theat least 4 contiguous nucleosides comprising 2′-hydroxyl pentofuranosylsugar moieties of the second chemically synthesized oligomeric compoundhybridize to each other in the duplex.
 95. (canceled)
 96. Thecomposition of claim 89 wherein at least one of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises at least one nucleoside comprising a 2′sugar modification selected from, alkoxy, amino-alkoxy, allyloxy,imidazolylalkoxy, polyethylene glycol, and methoxyethoxy.
 97. (canceled)98. The composition of claim 89 wherein each of the first chemicallysynthesized oligomeric compound and the second chemically synthesizedoligomeric compound comprises at least one 2′ sugar modificationselected from, alkoxy, amino-alkoxy, allyloxy, imidazolylalkoxy,polyethylene glycol, and methoxyethoxy.
 99. (canceled)
 100. (canceled)101. The composition of claim 89 wherein at least one of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound comprises at least two nucleosides eachcomprising a sugar comprising a 2′-fluoro.
 102. (canceled) 103.(canceled)
 104. (canceled)
 105. The composition of claim 89 wherein atleast one of the first chemically synthesized oligomeric compound andthe second chemically synthesized oligomeric compound comprises at leasttwo nucleosides each comprising a sugar comprising a 2′-OCH₃.
 106. Thecomposition of claim 105 wherein the 5′ terminal nucleoside of at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises a sugarcomprising a 2′-OCH₃.
 107. The composition of claim 89 wherein each ofthe first chemically synthesized oligomeric compound and the secondchemically synthesized oligomeric compound comprises at least twonucleosides comprising a sugar comprising a 2′-OCH₃.
 108. Thecomposition of claim 107 wherein the 5′ terminal nucleoside of at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises a sugarcomprising a 2′-OCH₃.
 109. The composition of claim 89 wherein at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises at least twonucleosides comprising different 2′ sugar modifications.
 110. Thecomposition of claim 89 wherein the first chemically synthesizedoligomeric compound comprises at least one nucleoside comprising a 2′sugar modification and the second chemically synthesized oligomericcompound comprises at least one nucleoside that comprises a different 2′sugar modification.
 111. The composition of claim 89 wherein at leastone of the first chemically synthesized oligomeric compound and thesecond chemically synthesized oligomeric compound comprises at least onenucleoside comprising a sugar surrogate.
 112. The composition of claim89 wherein each of the first chemically synthesized oligomeric compoundand the second chemically synthesized oligomeric compound comprises atleast one nucleoside comprising a sugar surrogate.
 113. The compositionof claim 89 wherein at least one of the first chemically synthesizedoligomeric compound and the second chemically synthesized oligomericcompound comprises at least one chemically modified internucleosidelinkage.
 114. The composition of claim 113 wherein at least onechemically modified internucleoside linkage is a phosphorothioatelinkage.
 115. The composition of claim 89 wherein each of the firstchemically synthesized oligomeric compound and the second chemicallysynthesized oligomeric compound comprises at least one chemicallymodified internucleoside linkage.
 116. The composition of claim 115wherein at least one chemically modified internucleoside linkage is aphosphorothioate linkage.